Methods for producing an immune response to tuberculosis

ABSTRACT

Methods for producing an immune response to  Mycobacterium tuberculosis  (Mtb) are disclosed herein. In several examples, the immune response is a protective immune response. In additional embodiments, methods are disclosed for preventing an infection with Mtb, or treating an infection with Mtb. Pharmaceutical compositions for the prevention and/or treatment of tuberculosis are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 12/282,865,filed Sep. 12, 2008, now U.S. Pat. No. 7,842,299, which is the U.S.national stage of International Application No. PCT/US2007/006472, filedMar. 14, 2007, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Application No.60/782,364, filed Mar. 14, 2006. The prior applications are incorporatedherein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support pursuantto Grant No. NIH-R01-AI48090 and Grant No. NIH NIAID HHSN266200400081CN01-AI-40081 from the National Institutes of Health. This invention wasalso made with support from the Department of Veteran's Affairs. TheUnited States government has certain rights in the invention.

FIELD

This application relates to the field of immunology, more specificallyto methods for the production of an immune response to tuberculosisantigens.

BACKGROUND

Mycobacteria are a genus of aerobic intracellular bacterial organismsthat, upon infection of a host, survive within endosomal compartments ofmonocytes and macrophages. Human mycobacterial diseases includetuberculosis (caused by M. tuberculosis), leprosy (caused by M. leprae),Bairnsdale ulcers (caused by M. ulcerans), and various infections causedby M. marinum, M. kansasii, M. scrofulaceum, M. szulgai, M. xenopi, M.fortuitum, M. chelonei, M. haemophilum and M. intracellulare (seeWolinsky, E., Chapter 37 in Microbiology: Including Immunology andMolecular Genetics, 3rd Ed., Harper & Row, Philadelphia, 1980).

One third of the world's population harbors M. tuberculosis and is atrisk for developing tuberculosis (TB). In immunocompromised patients,tuberculosis is increasing at a nearly logarithmic rate, and multidrugresistant strains are appearing. In addition, Mycobacterial strainswhich were previously considered to be nonpathogenic strains (e.g., M.avium) have now become major killers of immunosuppressed AIDS patients.Moreover, current Mycobacterial vaccines are either inadequate (such asthe BCG vaccine for M. tuberculosis) or unavailable (such as for M.leprae) (Kaufmann, S., Microbiol. Sci. 4:324-328, 1987; U.S. Congress,Office of Technology Assessment, The Continuing Challenge ofTuberculosis, pp. 62-67, OTA-H-574, U.S. Government Printing Office,Washington, D.C., 1993).

Mycobacterium tuberculosis (Mtb)-specific CD4⁺ and CD8⁺ T cells arecritical for the effective control of Mtb infection. In the mouse model,passive transfer of CD4⁺ T cells to sublethally irradiated animalsrenders them less susceptible to Mtb infection (Orme, J Immunol.140:3589-3593, 1988). Mice in which the gene(s) for CD4 (CD4^(−/−)) orfor MHC Class II molecules are disrupted as well as wild-type micedepleted of CD4⁺ T cells demonstrate increased susceptibility to Mtbinfection (Flory et al., J Leukoc Biol. 51:225-229, 1992). In humans,human immunodeficiency virus-infected individuals are exquisitelysusceptible to developing tuberculosis (TB) after exposure to Mtb,supporting an essential role for CD4⁺ T cells (Hirsch et al., J InfectDis. 180:2069-2073, 1999). CD8⁺ T cells are also important for effectiveT cell immunity (see Lazarevic and Flynn, Am J Respir Crit Care Med.166:1116-1121, 2002). In humans, Mtb-specific CD8⁺ T cells have beenidentified in Mtb-infected individuals and include CD8⁺ T cells that areboth classically HLA-Ia restricted (see, for example, Lewinsohn et al.,J Immunol. 165:925-930, 2000) and nonclassically restricted by theHLA-Ib molecules HLA-E (Lewinsohn et al., J Exp Med 187:1633-1640,1998). However, there are no vaccines or therapeutic strategies thateffectively induce an immune response, such as a CD8 response, to Mtb.Thus, a need remains for agents that can produce an immune response toMtb that can be used for treatment and/or protection from an Mtbinfection.

SUMMARY

Methods for producing an immune response to Mycobacterium tuberculosis(Mtb) are disclosed herein. Methods for treating an Mtb infection, orpreventing an Mtb infection in a subject, are also disclosed herein. TheMtb infection can be latent or active.

In several embodiments, the methods include administering to the subjecta therapeutically effective amount of a polypeptide, or a polynucleotideencoding the polypeptide, wherein the polypeptide comprises at least oneof the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. Inadditional embodiments, the methods include administering to the subjecta therapeutically effective amount of a polypeptide comprising at leastnine to twenty consecutive amino acids of at least one of the amino acidsequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12,wherein the nine to twenty consecutive amino acids specifically bindmajor histocompatibility complex (MHC) class I. In several examples, theimmune response is a protective immune response. In additionalembodiments, methods are disclosed for preventing an infection with Mtb,or treating an infection with Mtb.

Isolated polypeptides are described herein that include nine to twentyconsecutive amino acids of at least one of the amino acid sequences setforth as SEQ ID NOs: 1-12, wherein the nine to twenty consecutive aminoacids specifically bind major histocompatibility complex (MHC) class I,wherein the isolated polypeptide does not include any of the full lengthamino acid sequences set forth as SEQ ID NOs: 1-12. Nucleic acidsencoding these polypeptides, vectors including these nucleic acids, hostcells including these nucleic acids, and immunogenic compositionsincluding these polypeptides, nucleic acids and/or host cells are alsodisclosed. Pharmaceutical compositions including a therapeuticallyeffective amount of these polypeptides, nucleic acids, and/or host cellsare also described.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is two graphs showing the determination of human effector cellfrequencies ex vivo using the IFN-γ ELISPOT assay. Magneticbead-purified CD8⁺ T cells were cultured with DC (20,000/well) eitherinfected with Mtb (H37Rv, MOI=50) or pulsed with peptide poolrepresenting CFP10 (5 μg/ml each peptide; 15-mers overlap 11 aa) in anIFN-γ ELISPOT assay. Each responding T cell population was tested induplicate at four different cell concentrations. To determine theeffector cell frequency of antigen-specific T cells, the average numberof spots per well for each duplicate was plotted against the number ofresponder cells per well. Linear regression analysis was used todetermine the slope of the line, which represents the frequency ofantigen-specific T cells. The assay was considered positive (reflectingthe presence of a primed T cell response), if the binomial probabilityfor the number of spots was significantly different by experimental andcontrol assays.

FIG. 2 is a set of graphs showing ex vivo CD8⁺ T cell frequencies to Mtbantigens are associated with Mtb infection. As described above (see FIG.1), to determine ex vivo CD8⁺ T cell frequencies, autologous DC eitherinfected with Mtb or pulsed with cognate peptide pools were incubatedwith CD8⁺ T cells in an IFN-γ ELISPOT assay. Subjects without evidencefor Mtb infection, those with LTBI, and those with active TB (cultureconfirmed pulmonary tuberculosis) were evaluated. “Mtb Infected”includes those with latent tuberculosis (TB) infection and activetuberculosis. P values are noted where P=<0.05(Wilcoxon/Kruskal-Wallis).

FIGS. 3 a to 3 d are a set of digital images showing the definition ofAntigenic Specificity and HLA-Restriction (the characterization of Tcell clone D466 D6). For the results shown in FIGS. 3 a-3 c, to identifythe antigen and minimal epitope recognized by T cell clone, D466 D6,T-cells (5000 cells/well) were incubated with autologous LCL(20,000/well) and 5 μg/ml of antigen. IFN-γ was assessed by ELISPOTafter eighteen hours of co-culture. For the results presented in FIG. 3a, antigens consisted of peptide pools representing known CD4⁺ antigens,made up of 15 amino acid (aa) peptides overlapping by 11 aa. For theresults presented in FIG. 3 b, antigens consisted of individual 15 aaCFP10 peptides that together constitute the peptide pool. For theresults presented in FIG. 3 c, antigens consisted of individual nestedCFP10₁₋₁₅ peptides (10 aa, 9 aa or 8 aa), used to further map theepitope. For the results presented in FIG. 3 d, the restricting allelewas identified using LCL (20,000/well) expressing HLA alleles matchingD466 at one or two alleles, pulsed with CFP10₂₋₁₀ (5 μg/ml) as APC.After 2 hours, cells were washed and incubated with T-cells (500cells/well) in an IFN-γ ELISPOT assay.

FIG. 4 is a line graph showing the confirmation of minimal epitopemapping of D466 D6. The epitope location (amino acids 1-15, 2-12, 3-13,2-11 and 3-12 of CFP10) is indicated in the legend shown at the right.To confirm the minimal epitope, autologous LCL (20,000/well) was pulsedwith peptide at the concentration indicated and co-cultured with T-cells(1000 cells/well). IFN-γ was assessed by ELISPOT after eighteen hoursco-culture. Each point represents the mean of duplicate determinations.

FIG. 5 is a set of bar graphs showing the profiling of theimmunodominance pattern for CFP10. To determine the effector cellfrequencies, autologous DC (20,000/well) were pulsed either with eachindividual 15-mer peptide (5 μg/ml), the peptide pool (PP; 5 μg/eachpeptide) or the minimal epitope (ME) determined from T cell clonesderived from each donor (D466:CFP10₂₋₁₁; D480:CFP10₃₋₁₁;D481:CFP10₇₅₋₈₃; 5 μg/ml), and tested against 250,000 magnetic beadpurified CD8⁺ T cells. IFN-γ release was assessed by ELISPOT aftereighteen hours of co-culture. Each point represents the mean ofduplicate determinations.

FIGS. 6 a and 6 b are sets of graphs summarizing the minimal epitopemapping data. To determine the minimal epitope, autologous LCL(20,000/well) was pulsed with peptide at the concentration indicated andco-cultured with T-cells (1000 cells/well). IFN-γ was assessed byELISPOT after eighteen hours co-culture. Each point represents the meanof duplicate determinations.

FIG. 7 is a line graph showing the mapping of Minimal Epitope for D504Clones. To determine the minimal epitope, autologous LCL (20,000/well)was co-cultured with T-cell clones (1,000 cells/well) and the peptide atthe concentration indicated. IFN-γ was assessed by ELISPOT aftereighteen hours co-culture. Each point represents the mean of duplicatedeterminations. The spot forming units for peptides with amino acids21-35, 23-33, 24-34, 24-33 and 24-32 are shown.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

The Sequence Listing is submitted as an ASCII text file[899-77364-19-09_Sequence_Listing.txt, Sep. 29, 2010, 84.4 KB], which isincorporated by reference herein.

SEQ ID NOs: 1-12 are the amino acid sequence of Mtb polypeptides.

SEQ ID NOs: 13-14 are amino acids of Mtb peptides.

SEQ ID NOs: 15-25 are the nucleic acid sequences of polynucleotidesencoding the Mtb polypeptides.

SEQ ID NOs: 26-38 are the amino acid sequences of specific Mtb epitopes.

DETAILED DESCRIPTION

Methods for producing an immune response to Mycobacterium tuberculosis(Mtb) are disclosed herein. In several examples, the immune response isa protective immune response. In additional embodiments, methods aredisclosed for preventing an infection with Mtb, or treating an infectionwith Mtb. Pharmaceutical compositions for the prevention and/ortreatment of tuberculosis are also disclosed.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN¹-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include asuspension of minerals (alum, aluminum hydroxide, or phosphate) on whichantigen is adsorbed; or water-in-oil emulsion in which antigen solutionis emulsified in mineral oil (Freund incomplete adjuvant), sometimeswith the inclusion of killed mycobacteria (Freund's complete adjuvant)to further enhance antigenicity (inhibits degradation of antigen and/orcauses influx of macrophages). Immunostimulatory oligonucleotides (suchas those including a CpG motif) can also be used as adjuvants (forexample see U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants includebiological molecules (a “biological adjuvant”), such as costimulatorymolecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Generally, T cellsrecognize epitopes of continuous amino acids. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue. Atissue specific antigen may be expressed by more than one related typeof tissue, such as alveolar and bronchial tissue. A disease-specificantigen is expressed coincidentally with a disease process. Specificnon-limiting examples of a disease-specific antigen are an antigen whoseexpression correlates with, or is predictive of, tuberculosis. Adisease-specific antigen can be an antigen recognized by T cells or Bcells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the V_(L), V_(H),C_(L) and C_(H1) domains; (ii) an F_(d) fragment consisting of the V_(H)and C_(H1) domains; (iii) an Fv fragment consisting of the V_(L) andV_(H) domains of a single arm of an antibody, (iv) a dAb fragment (Wardet al., Nature 341:544-546, 1989) which consists of a V_(H) domain; (v)an isolated complementarity determining region (CDR); and (vi) a F(ab)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. Nos.4,745,055; 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023;Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793,1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antigen presenting cell (APC): A cell that can present an antigen to a Tcell, such that the T cells are activated. Dendritic cells are theprinciple antigen presenting cells (APCs) involved in primary immuneresponses. Their major function is to obtain antigen in tissues, migrateto lymphoid organs and present the antigen in order to activate T cells.

When an appropriate maturational cue is received, dendritic cells aresignaled to undergo rapid morphological and physiological changes thatfacilitate the initiation and development of immune responses. Amongthese are the up-regulation of molecules involved in antigenpresentation; production of pro-inflammatory cytokines, including IL-12,key to the generation of Th1 responses; and secretion of chemokines thathelp to drive differentiation, expansion, and migration of surroundingnaive Th cells. Collectively, these up-regulated molecules facilitatethe ability of dendritic cells to coordinate the activation and effectorfunction of other surrounding lymphocytes that ultimately provideprotection for the host.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells. “CD8+ T cell mediated immunity” is animmune response implemented by presentation of antigens to CD8+ T cells.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of the Mycobacterium polypeptide. Specific, non-limitingexamples of a conservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide, or that an immune response can begenerated against the substituted polypeptide that is similar to theimmune response against and unsubstituted polypeptide, such aMycobacterium antigen. Thus, in one embodiment, non-conservativesubstitutions are those that reduce an activity or antigenicity.

Consists Essentially Of/Consists Of: With regard to a polypeptide, apolypeptide that consists essentially of a specified amino acid sequenceif it does not include any additional amino acid residues. However, thepolypeptide can include additional non-peptide components, such aslabels (for example, fluorescent, radioactive, or solid particlelabels), sugars or lipids. With regard to a polypeptide, a polypeptidethat consists of a specified amino acid sequence does not include anyadditional amino acid residues, nor does it include additionalnon-peptide components, such as lipids, sugars or labels.

Contacting: The process of incubating one agent in the presence ofanother. Thus, when a cell is contacted with an agent, the cell isincubated with the agent for a sufficient period of time for the agentand the cell to interact.

Costimulatory molecule: Although engagement of the TCR with peptide-MHCdelivers one signal to the T cell, this signal alone can be insufficientto activate the T cell. Costimulatory molecules are molecules that, whenbound to their ligand, deliver a second signal required for the T cellto become activated. The most well-known costimulatory molecule on the Tcell is CD28, which binds to either B7-1 (also called CD80) or B7-2(also known as CD86). An additional costimulatory molecule is B7-3.Accessory molecules that also provide a second signal for the activationof T cells include intracellular adhesion molecule (ICAM-1 and ICAM-2),and leukocyte function associated antigen (LFA-1, LFA-2 and LFA-3).Integrins and tumor necrosis factor (TNF) superfamily members can alsoserve as co-stimulatory molecules.

Cytokine: Proteins made by cells that affect the behavior of othercells, such as lymphocytes. In one embodiment, a cytokine is achemokine, a molecule that affects cellular trafficking. Specific,non-limiting examples of cytokines include the interleukins (IL-2, IL-4,IL-6, IL-10, IL-21, etc.), and IFN-γ.

Degenerate variant: A polynucleotide encoding an epitope of an Mtbpolypeptide that includes a sequence that is degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in this disclosure as long as the amino acidsequence of the Mtb polypeptide encoded by the nucleotide sequence isunchanged.

Dendritic cell (DC): Dendritic cells are the principle antigenpresenting cells (APCs) involved in primary immune responses. Dendriticcells include plasmacytoid dendritic cells and myeloid dendritic cells.Their major function is to obtain antigen in tissues, migrate tolymphoid organs and present the antigen in order to activate T cells.Immature dendritic cells originate in the bone marrow and reside in theperiphery as immature cells.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, tuberculosis. Diagnostic methodsdiffer in their sensitivity and specificity. The “sensitivity” of adiagnostic assay is the percentage of diseased individuals who testpositive (percent of true positives). The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the false positive rateis defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” meanspredicting the probability of development (for example, severity) of apathologic condition, such as tuberculosis.

Displaying: The process of localizing a peptide:antigen complex, or apeptide, on the outer surface of a cell where the peptide:antigencomplex or peptide is accessible to a second cell, molecules displayedby a second cell, or soluble factors. A peptide, or a peptide:antigencomplex, is “displayed” by a cell when it is present on the outersurface of the cell and is accessible to a second cell, to moleculesdisplayed by the second cell, or to soluble factors.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such a Mycobacterium polypeptide.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters, are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences. In one embodiment, thepromoter is a cytomegalovirus promoter.

Fractionating: Subjecting a sample to conditions or procedures whichseparate the components of the sample based on physical or chemicalproperties such as, but not limited to, size, charge, solubility, orcomposition. Example of fractionation procedures include, but are notlimited to, selective precipitation, organic extraction, size exclusiondialysis or chromatography, such as ion exchange chromatography. In oneembodiment, a fraction is a soluble extract or an organic extract of anorganism, such as a Mycobacterium.

Functionally Equivalent Sequence alterations, such as in an epitope ofan antigen, that yield the same results as described herein. Suchsequence alterations can include, but are not limited to, conservativesubstitutions, deletions, mutations, frameshifts, and insertions.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to an Mtb polypeptide originates from anucleic acid that does not encode the Mtb polypeptide. In one specific,non-limiting example, a polypeptide comprising nine consecutive aminoacids from an Mtb polypeptide, or at most 20 consecutive amino acidsfrom the Mtb polypeptide, and a heterologous amino acid sequenceincludes a β-galactosidase, a maltose binding protein, albumin,hepatitis B surface antigen, or an immunoglobulin amino acid sequence.Generally, an antibody that specifically binds to a protein of interestwill not specifically bind to a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Human Leukocyte Antigen (HLA): A genetic designation of the human majorhistocompatibility complex (MHC). Individual loci are designated byuppercase letters, as in HLA-E, and alleles are designated by numbers,as in HLA-A*0201. The three main MHC class I genes are called HLA-A,HLA-B, and HLA-C. However, there are many genes that encode 132microglobulin-associated cell surface molecules that are linked to theMHC class I genes. The expression of these genes is variable, both inthe tissue distribution and the amount expressed on cells; these geneshave been termed the MHC class IB genes.

Immune response: A response of a cell of the immune system, such as a Bcell, natural killer cell, or a T cell, to a stimulus. In oneembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In one embodiment, an immune response is aT cell response, such as a Th1, Th2, or Th3 response. In anotherembodiment, an immune response is a response of a suppressor T cell.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen from which theimmunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their bindingto a specific MHC protein and by their ability to stimulate CD4 and/orCD8 when presented in the context of the MHC protein.

In one example, an immunogenic “Mtb peptide” is a series of contiguousamino acid residues from the Mtb protein generally between 9 and 20amino acids in length, such as about 9 to 12 residues in length.Specific immunogenic polypeptides are disclosed herein that are 9 or 10amino acid residues in length, or at most 12 amino acids in length.Generally, immunogenic Mtb polypeptides can be used to induce an immuneresponse in a subject, such as a B cell response or a T cell response.In one example, an immunogenic Mtb polypeptide, when bound to a MHCClass I molecule, activates cytotoxic T lymphocytes (CTLs) against Mtb.Induction of CTLs using synthetic peptides and CTL cytotoxicity assaysare known in the art, see U.S. Pat. No. 5,662,907, which is incorporatedherein by reference. In one example, an immunogenic peptide includes anallele-specific motif or other sequence such that the peptide will bindan MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) responseagainst the antigen from which the immunogenic peptide is derived.

Immunogenic composition: A composition comprising an immunogenic Mtbpolypeptide or a nucleic acid encoding the immunogenic Mtb polypeptidethat induces a measurable CTL response against Mtb, or induces ameasurable B cell response (such as production of antibodies thatspecifically bind an Mtb polypeptide). For in vitro use, the immunogeniccomposition can consist of the isolated nucleic acid, vector includingthe nucleic acid/or immunogenic peptide. For in vivo use, theimmunogenic composition will typically comprise the nucleic acid, vectorincluding the nucleic acid, and or immunogenic polypeptide, inpharmaceutically acceptable carriers, and/or other agents. Animmunogenic composition can optionally include an adjuvant, acostimulatory molecule, or a nucleic acid encoding a costimulatorymolecule. An Mtb polypeptide, or nucleic acid encoding the polypeptide,can be readily tested for its ability to induce a CTL by art-recognizedassays.

Inhibiting or treating a disease: Inhibiting a disease, such astuberculosis, refers to inhibiting the full development of a disease. Inseveral examples, inhibiting a disease refers to lessening symptoms of atuberculosis. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionrelated to the disease, such as tuberculosis.

Interferon gamma (γ): IFN-γ is a dimeric protein with subunits of 146amino acids. The protein is glycosylated at two sites, and the pI is8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kDa have beendescribed. Both of them are glycosylated at position 25. The 25 kDa formis also glycosylated at position 97. The observed differences of naturalIFN-γ with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELISPOT testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasbeen used also to measure IFN-γ concentrations. In addition, bioassayscan be used to detect IFN-γ, such as an assay that employs induction ofindoleamine 2,3-dioxygenase activity in 2D9 cells. The production ofIFN-γ can be used to assess T cell activation, such as activation of a Tcell by an HLA-E presented Mycobacterium antigen.

Isolated: An “isolated” nucleic acid has been substantially separated orpurified away from other nucleic acid sequences in the cell of theorganism in which the nucleic acid naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA. The term “isolated” thusencompasses nucleic acids purified by standard nucleic acid purificationmethods. The term also embraces nucleic acids prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in between the Mtb epitopes disclosed herein to providerotational freedom to the linked polypeptide domains and thereby topromote proper domain folding and presentation to the MHC. By way ofexample, in a recombinant polypeptide comprising two Mtb domains, linkersequences can be provided between them, such as a polypeptide comprisingMtb polypeptide-linker-Mtb polypeptide. Linker sequences, which aregenerally between 2 and 25 amino acids in length, are well known in theart and include, but are not limited to, the glycine(4)-serine spacer(GGGGS x3) described by Chaudhary et al., Nature 339:394-397, 1989.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “patient” or “subject” includes both human and veterinarysubjects.

Mycobacteria: A genus of aerobic intracellular bacterial organisms. Uponinvasion of a host, these organisms survive within endosomalcompartments of monocytes and macrophages. Human mycobacterial diseasesinclude tuberculosis (cause by M. tuberculosis), Leprosy (caused by M.leprae), Bairnsdale ulcers (caused by M. ulcerans), and other infectionsthat can be caused by M. marinum, M. kansasii, M. scrofulaceum, M.szulgai, M. xenopi, M. fortuitum, M. haemophilum, M. chelonei, and M.intracelluare. Mycobacterium strains that were previously considered tobe nonpathogenic (such as M. avium) are also now known to be majorkillers of immunosuppressed AIDS patients.

An immune response to mycobacteria can involve cell mediatedhypersensitivity (DTH) reactions with T cells and macrophages playingmajor roles in the intracellular killing and walling off (or containing)of the organism (granuloma formation). A T cell response can involveCD4⁺ lymphocytes that recognize myocbacterial heat shock proteins andimmunodominant antigens as well as CD8⁺ cells.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter effects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, the openreading frames are aligned.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a polypeptide.

Peptide Modifications: Mycobacterium polypeptides include syntheticembodiments of peptides described herein. In addition, analogues(non-peptide organic molecules), derivatives (chemically functionalizedpeptide molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of these proteins can be utilized inthe methods described herein. Each polypeptide of the invention iscomprised of a sequence of amino acids, which may be either L- and/orD-amino acids, naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of a Mycobacterium polypeptide havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful with the polypeptides and nucleic acids described hereinare conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,Mack Publishing Co., Easton, Pa., 15th Edition (1975), describescompositions and formulations suitable for pharmaceutical delivery ofthe fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: A linear nucleotide sequence, including sequences ofgreater than 100 nucleotide bases in length.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). A “peptide” is a chain of amino acids that is lessthan 100 amino acids in length. In one embodiment, a “peptide” is aportion of a polypeptide, such as at about 10, 20, 30, 40, 50, or 100contiguous amino acids of a polypeptide that is greater than 100 aminoacids in length.

Portion of a nucleic acid sequence: At least 10, 20, 30 or 40 contiguousnucleotides of the relevant sequence, such as a sequence encoding anantigen. In some instances it would be advantageous to use a portionconsisting of 50 or more nucleotides. For instance, when describing aportion of an antigen (such as an antigenic epitope), it may beadvantageous to remove a portion of the relevant sequence comprising atleast 10, 20, 30, 40 or 50 nucleotides up to a length.

Probes and primers: Nucleic acid probes and primers may readily beprepared based on the nucleic acids provided by this invention. A probecomprises an isolated nucleic acid attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al. (1989) and Ausubel et al. (1987).

Primers are short nucleic acids, preferably DNA oligonucleotides 15nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, and Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1987(with periodic updates). PCR primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer (Version 0.5, © 1991, Whitehead Institute forBiomedical Research, Cambridge, Mass.).

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example in a personwho is known to be at risk of infection with M. tuberculosis, or M.leprae. An example of a person with a known predisposition is someoneliving with a person diagnosed with tuberculosis, health careprofessionals, or someone otherwise known to have been exposed to M.tuberculosis. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological condition,such as tuberculosis, after it has begun to develop.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription. The promoter can be a constitutive or an induciblepromoter. A specific, non-limiting example of a promoter is the HCMV IEpromoter.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified antigenpreparation is one in which the antigen is more pure than the protein inits originating environment within a cell. A preparation of an antigenis typically purified such that the antigen represents at least 50% ofthe total protein content of the preparation. However, more highlypurified preparations may be required for certain applications. Forexample, for such applications, preparations in which the antigencomprises at least 75% or at least 90% of the total protein content maybe employed.

Recombinant: A recombinant nucleic acid or polypeptide is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two or more otherwise separated segmentsof sequence. This artificial combination is often accomplished bychemical synthesis or, more commonly, by the artificial manipulation ofisolated segments of nucleic acids, e.g., by genetic engineeringtechniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Variants of antigen polypeptides will possess a relatively high degreeof sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Altschul et al. (1994) presents a detailed consideration ofsequence alignment methods and homology calculations. The NCBI BasicLocal Alignment Search Tool (BLAST) (Altschul et al., 1990) is availablefrom several sources, including the National Center for BiotechnologyInformation (NCBI, Bethesda, Md.) and on the Internet, for use inconnection with the sequence analysis programs blastp, blastn, blastx,tblastn and tblastx. It can be accessed at the NCBI website. Adescription of how to determine sequence identity using this program isavailable at the NCBI website, as are the default parameters.

Variants of antigenic polypeptides, such as a Mycobacterium polypeptide,are typically characterized by possession of at least 50% sequenceidentity counted over the full length alignment with the amino acidsequence of a native antigen sequence using the NCBI Blast 2.0, gappedblastp set to default parameters. Proteins with even greater similarityto the reference sequences will show increasing percentage identitieswhen assessed by this method, such as at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 90% or at least 95%sequence identity. When less than the entire sequence is being comparedfor sequence identity, variants will typically possess at least 75%sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85% or at least 90% or 95%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI website. Variants of MHC domain polypeptides also retain thebiological activity of the native polypeptide.

Therapeutically active polypeptide: An agent, such as an epitope of Mtbthat causes induction of an immune response, as measured by clinicalresponse (such as an increase in a population of immune cells, increasedcytolytic activity against Mtb, or measurable reduction of a symptom ofan infection). Therapeutically active molecules can also be made fromnucleic acids. Examples of a nucleic acid based therapeutically activemolecule is a nucleic acid sequence that encodes a Mtb epitope, whereinthe nucleic acid sequence is operably linked to a control element suchas a promoter.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease. In one embodiment, atherapeutically effective dose is a dose sufficient to preventadvancement or relieve symptoms of tuberculosis.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” by anucleic acid transduced into the cell when the DNA becomes stablyreplicated by the cell, either by incorporation of the nucleic acid intothe cellular genome, or by episomal replication. As used herein, theterm transformation encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Tuberculosis (TB): A disease that is generally caused by Mycobacteriumtuberculosis that usually infects the lungs. However, other “atypical”mycobacteria such as M. kansasii may produce a similar clinical andpathologic appearance of disease.

Transmission of M. tuberculosis occurs by the airborne route in confinedareas with poor ventilation. In more than 90% of cases, followinginfection with M. tuberculosis, the immune system prevents developmentof disease from M. tuberculosis, often called, active tuberculosis.However, not all of the M. tuberculosis is killed, and thus tiny, hardcapsules are formed. “Primary tuberculosis” is seen disease thatdevelops following an initial infection, usually in children. Theinitial focus of infection is a small subpleural granuloma accompaniedby granulomatous hilar lymph node infection. Together, these make up theGhon complex. In nearly all cases, these granulomas resolve and there isno further spread of the infection. “Secondary tuberculosis” is seenmostly in adults as a reactivation of previous infection (orreinfection), particularly when health status declines. Thegranulomatous inflammation is much more florid and widespread.Typically, the upper lung lobes are most affected, and cavitation canoccur. Dissemination of tuberculosis outside of lungs can lead to theappearance of a number of uncommon findings with characteristic patternsthat include skeletal tuberculosis, genital tract tuberculosis, urinarytract tuberculosis, central nervous system (CNS) tuberculosis,gastrointestinal tuberculosis, adrenal tuberculosis, scrofula, andcardiac tuberculosis. “Latent” tuberculosis is an Mtb infection in anindividual that can be detected by a diagnostic assay, such as, but notlimited to a tuberculin skin test (TST) wherein the infection does notproduce symptoms in that individual. “Active” tuberculosis is asymptomatic Mtb infection in a subject.

Microscopically, the inflammation produced with TB infection isgranulomatous, with epithelioid macrophages and Langhans giant cellsalong with lymphocytes, plasma cells, maybe a few polymorphonuclearcells, fibroblasts with collagen, and characteristic caseous necrosis inthe center. The inflammatory response is mediated by a type IVhypersensitivity reaction, and skin testing is based on this reaction.In some examples, tuberculosis can be diagnosed by a skin test, an acidfast stain, an auramine stain, or a combination thereof. The most commonspecimen screened is sputum, but the histologic stains can also beperformed on tissues or other body fluids.

TB is a frequent complication of HIV infection. TB infection in subjectsinfected with a human immunodeficiency virus (HIV) can spread readilyand progress rapidly to active disease. Specific symptoms of lungdisease due to Mtb infection include chronic cough and spitting blood.Other symptoms of TB disease include fatigue, loss of appetite, weightloss, fever and drenching night sweats.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transduced or transformed host cell. A vector may includenucleic acid sequences that permit it to replicate in a host cell, suchas an origin of replication. A vector may also include one or moreselectable marker gene and other genetic elements known in the art.Vectors include plasmid vectors, including plasmids for expression ingram negative and gram positive bacterial cells. Exemplary vectorsinclude those for expression in E. coli and Salmonella. Vectors alsoinclude viral vectors, such as, but are not limited to, retrovirus,orthopox, avipox, fowlpox, capripox, suipox, adenoviral, herpes virus,alpha virus, baculovirus, Sindbis virus, vaccinia virus and poliovirusvectors. Vectors also include vectors for expression in yeast cells.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Mycobacterium Polypeptides

It is disclosed herein that several Mycobacterium polypeptides can beused to induce an immune response to Mtb, such as a T cell response. Inseveral embodiments, the polypeptide comprises or consists of the aminoacid sequence set forth as:

1. MX1SRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMX₂X₃MNQAFRNIVNMLHGVRDGLVRDANNY EQQEQASQQILS, (SEQ ID NO: 1, wherein X1 is A or T, X₂ is T or A and X₃ is any amino acid,such as Q  or no amino acid)In several examples, the polypeptide comprises or consists of the aminoacid sequence set forth as:

a. MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANNYEQ QEQASQQILS (SEQ ID NO: 2) (See also TUBERCULIST No. Rv1038c, as available on Mar. 1, 2007,incorporated herein by reference, known as EsxJ, ES6_2, TB11.0, QILSS)b. MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMAQMNQAFRNIVNMLHGVRDGLVRDANNYE QQEQASQQILSS (SEQ ID NO: 3, TUBERCULIST No. Rv1197, asavailable on Mar. 1, 2007, incorporated herein by reference, also know as EsxK, ES6_3, TB11.0, QILSS)c. MASRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISG AGWSGMAEATSLDTMT +MNQAFRNIVNMLHGVRDGLVRDANNYE QQEQASQQILSS (SEQ ID NO: 4, TUBERCULIST No. Rv 1992,as available on Mar. 1, 2007, incorporatedherein by reference, as known as EsxM,  TB11.0, QILSS.d. MATRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMAQMNQAFRNIVNMLHGVRDGLVRDANNYE QQEQASQQILSS (SEQ ID NO: 5, TUBERCULIST No. Rv 2347c,as available on Mar. 1, 2007, incorporatedherein by reference, also known as EsxP, ES6_7, QILSS)e. MTSRFMTDPHAMRDMAGRFEVHAQTVEDEARRMWASAQNISGAGWSGMAEATSLDTMTQMNQAFRNIVNMLHGVRDGLVRDANNYE QQEQASQQILSS (SEQ ID NO: 6, TUBERCULIST No. Rv3620c,as available on Mar. 1, 2007, incorporatedherein by reference, also known as EsxW, ES6_10, QILSS).In additional embodiments, the polypeptide comprises or consists of theamino acid sequence set forth as:

2. MSYMIATPAALTAAATDIDGIGSAVSVANAAAVAATTGVLAAGGDEVLAAIARLFNANAEEYHALSAQVAAFQTLFVRTLTGGCGVFRRRRGRQCVTAAEHRAAGAGRRQRRRRSGDGQW RLRQQRHFGCGGQPEFRQHSEHRR(SEQ ID NO: 7, TUBERCULIST NO. Rv1088, asavailable on Mar. 1, 2007, incorporatedherein by reference, also known as PE9).3. VSLVIATPQLLATAALDLASIGSQVSAANAAAAMPTTEVVAAAADEVSAAIAGLFGAHARQYQALSVQVAAFHEQFVQALTAAAGRYASTEAAVERSLLGAVNAPTEALLGRPLIGNGADGTAPGQPGAAGGLLFGNGGNGAAGGFGQTGGSGGAAGLIGNGGNGGAGGTGAAGGAGGNGGWLWGNGGNGGVGGTSVAAGIGGAGGNGGNAGLFGHGGAGGTGGAGLAGANGVNPTPGPAASTGDSPADVSGIGDQTGGDGGTGGHGTAGTPTGGTGGDGATATAGSGKATGGAGGDGGTAAAGGGGGNGGDGGVAQGDIASAFGGDGGNGSDGVAAGSGGGSGGAGGGAFVHIATATSTGGSGGFGGNGAASAASGADGGAGGAGGNGGAGGLLFGDGGNGGAGGAGGIGGDGATGGPGGSGGNAGIARFDSPDPEAEPDVVGGKGGDGGKGGSGLGVGGAGGTGGAGGNGGAGGLLFGNGGNGGNAGAGGDGGAGVAGGVGGNGGGGGTATFHEDPVAGVWAVGGVGGDGGSGGSSLGVGGVGGAGGVGGKGGASGMLIGNGGNGGSGGVGGAGGVGGAGGDGGNGGSGGNASTFGDENSIGGAGGTGGNGGNGANGGNGGAGGIAGGAGGSGGFLSGAAGVSGADGIGGAGGAGGAG GAGGSGGEAGAGGLTNGPGSPGVSGTEGMAGAPG(SEQ ID NO: 8, TUBERCULIST NO. Rv2487, asavailable on Mar. 1, 2007, incorporatedherein by reference, also known as PE_PGRS42)4. MHQVDPNLTRRKGRLAALAIAAMASASLVTVAVPATANADPEPAPPVPTTAASPPSTAAAPPAPATPVAPPPPAAANTPNAQPGDPNAAPPPADPNAPPPPVIAPNAPQPVRIDNPVGGFSFALPAGWVESDAAHFDYGSALLSKTTGDPPFPGQPPPVANDTRIVLGRLDQKLYASAEATDSKAAARLGSDMGEFYMPYPGTRINQETVSLDANGVSGSASYYEVKFSDPSKPNGQIWTGVIGSPAANAPDAGPPQRWFVVWLGTANNPVDKGAAKALAESIRPLVAPPPAPAPAPAEP APAPAPAGEVAPTPTTPTPQRTLPA(SEQ ID NO: 9, TUBERCULIST No. Rv1860, asavailable on Mar. 1, 2007, incorporated hereinby reference, also known as Apa, modD, mpt32)5. MLLALLRQHIRPYRRLVAMLMMLQLVSTLASLYLPTVNAAIVDDGVAKGDTATIVRLGAVMLGVTGLQVLCAIGAVYLGSRTGAGFGRDLRSAMFEHIITFSERETARFGAPTLLTRSTNDVRQILFLVQMTATVLVTAPIMCVGGIIMAIHQEAALTWLLLVSVPILAVANYWIISHMLPLFRRMQSLIDGINRVMRDQLSGVRVVRAFTREGYERDKFAQANTALSNAALSAGNWQALMLPVTTLTINASSVALIWFGGLRIDSGQMQVGSLIAFLSYFAQILMAVLMATMTLAVLPRASVCAERITEVLSTPAALGNPDNPKFPTDGVTGVVRLAGATFTYPGADCPVLQDISLTARPGTTTAIVGSTGSGKSTLVSLICRLYDVTAGAVLVDGIDVREYHTERLWSAIGLVPQRSYLFSGTVADNLRYGGGPDQVVTEQEMWEALRVAAADGFVQTDGLQTRVAQGGVNFSGGQRQRLAIARAVIRRPAIYVFDDAFSALDVHTDAKVHASLRQVSGDATIIVVTQRISNAAQADQVIVVDNGKIVGTGTHETL LADCPTYAEFAASQSLSATVGGVG(SEQ ID NO: 10, TUBERCULIST NO. Rv1273c, asavailable Mar. 1, 2007, incorporated herein by reference).6. MSYVIAAPEMLATTAADVDGIGSAIRAASASAAGPTTGLLAAAADEVSSAAAALFSEYARECQEVLKQAAAFHGEFTRALAAAGAAYAQAEASNTAAMSGTAGSSGALGSVGMLSGNPLTALMMGGTGEPILSDRVLAIIDSAYIRPIFGPNNPVAQYTPEQWWPFIGNLSLDQSIAQGVTLLNNGINAELQNGHDVVVFGYSQSAAVATNEIRALMALPPGQAPDPSRLAFTLIGNINNPNGGVLERYVGLYLPFLDMSFNGATPPDSPYQTYMYTGQYDGYAHNPQYPLNILSDLNAFMGIRWVHNAYPFTAAEVANAVPLPTSPGYTGNTHYYMFLTQDLPLLQPIRAIPFVGTPIAELIQPDLRVLVDLGYGYGYADVPTPASLFAPINPIAVASALATGTVQGPQAALVSIGLLPQSALPNTYPYLPSANPGLMFNFGQSSVTELSVLSGALGSVARLIPPIA(SEQ ID NO: 11, TUBERCULIST NO. Rv0159c, asavailable Mar. 1, 2007, incorporated herein byreference, also know as PE3 or PE).7. MEFPVLPPEINSVLMYSGAGSSPLLAAAAAWDGLAEELGSAAVSFGQVTSGLTAGVWQGAAAAAMAAAAAPYAGWLGSVAAAAEAVAGQARVVVGVFEAALAATVDPALVAANRARLVALAVSNLLGQNTPAIAAAEAEYELMWAADVAAMAGYHSGASAAAAALPAFSPPAQALGGGVGAFLTALFASPAKALSLNAGLGNVGNYNVGLGNVGVFNLGAGNVGGQNLGFGNAGGTNVGFGNLGNGNVGFGNSGLGAGLAGLGNIGLGNAGSSNYGFANLGVGNIGFGNTGTNNVGVGLTGNHLTGIGGLNSGTGNIGLFNSGTGNVGFFNSGTGNFGVFNSGNYNTGVGNAGTASTGLFNAGNFNTGVVNVGSYNTGSFNAGDTNTGGFNPGGVNTGWLNTGNTNTGIANSGNVNTGAFISGNFNNGVLWVGDYQGLFGVSAGSSIPAIPIGLVLNGDIGPITIQPIPILPTIPLSIHQTVNLGPLVVPDIVIPAFGGGIGIPINIGPLTITPITLFAQQTFVNQLPFPTFSLGKTTIPQIQTFDSNGQLVSFIGPIVIDTTIPGPTNPQIDLTIRWDTPPITLFPNGISAPDNPLGLLVSVSISNPGFTIPGFSVPAQPLPLSIDIEGQIDGFSTPPITIDRIPLTVGGGVTIGPITIQGLHIPAAPGVGNYITAPSSGFFNSGAGGVSGFGNVGAGSSGWWNQAPSALLGAGSGVGNVGTLGSGVLNLGSGISGFYNTSVLPFGTPAAVSGIGNLGQQLSGVSAAGTTLRSMLAGNLGLANVGNFNTGFGNVGDVNLGAANIGGHNLGLGNVGDGNLGLGNIGHGNLGFANLGLTAGAAGVGNVGFGNAGINNYGLANMGVGNIGFANTGTGNIGIGLVGDHRTGIGGLNSGIGNIGLFNSGTGNVGFFNSGTGNFGIGNSGRFNTGIGNSGTASTGLFNAGSFSTGIANTGDYNTGSFNAGDTNTGGFNPGGINTGWFNTGHANTGLANAGTFGTGAFMTGDYSNGLLWRGGYEGLVGVRVGPTISQFPVTVHAIGGVGPLHVAPVPVPAVHVEITDATVGLGPFTVPPISIPSLPIASITGSVDLAANTISPIRALDPLAGSIGLFLEPFRLSDPFITIDAFQVVAGVLFLENIIVPGLTVSGQILVTPTPIPLTLNLDTTPWTLFPNGFTIPAQTPVTVGMEVANDGFTFFPGGLTFPRASAGVTGLSVGLDAFTLLPDGFTLDTVPATFDGTILIGDIPIPIIDVPAVPGFGNTTTAPSSGFFNTGGGGGSGFANVGAGTSGWWNQGHDVLAGAGSGVANAGTLSSGVLNVGSGISGWYNTSTLGAGTPAVVSGIGNLGQQLSGFLANGTVLNRSPIVNIGWADVGAFNTGLGNVGDLNWGAANIGAQNLGLGNLGSGNVGFGNIGAGNVGFANSGPAVGLAGLGNVGLSNAGSNNWGLANLGVGNIGLANTGTGNIGIGLVGDYQTGIGGLNSGSGNIGLFNSGTGNVGFFNTGTGNFGLFNSGSFNTGIGNSGTGSTGLFNAGNFNTGIANPGSYNTGSFNVGDTNTGGFNPGDINTGWFNTGIMNTGTRNTGALMSGTDSNGMLWRGDHEGLFGLSYGITIPQFPIRITTTGGIGPIVIPDTTILPPLHLQITGDADYSFTVPDIPIPAIHIGINGVVTVGFTAPEATLLSALKNNGSFISFGPITLSNIDIPPMDFTLGLPVLGPITGQLGPIHLEPIVVAGIGVPLEIEPIPLDAISLSESIPIRIPVDIPASVIDGISMSEVVPIDASVDTPAVTITGTTISAIPLGFDIRTSAGPLNIPIIDIPAAPGFGNSTQMPSSGFFNTGAGGGSGIGNLGAGVSGLLNQAGAGSLVGTLSGLGNAGTLASGVLNSGTAISGLFNVSTLDATTPAVISGFSNLGDHMSGVSIDGLIAILTFPPAESVFDQIIDAAIAELQHLDIGNALALGNVGGVNLGLANVGEFNLGAGNVGNINVGAGNLGGSNLGLGNVGTGNLGFGNIGAGNFGFGNAGLTAGAGGLGNVGLGNAGSGSWGLANVGVGNIGLANTGTGNIGIGLTGDYRTGIGGLNSGTGNLGLFNSGTGNIGFFNTGTGNFGLFNSGSYSTGVGNAGTASTGLFNAGNFNTGLANAGSYNTGSLNVGSFNTGGVNPGTVNTGWFNTGHTNTGLFNTGNVNTGAFNSGSFNNGALWTGDYHGLVGFSFSIDIAGSTLLDLNETLNLGPIHIEQIDIPGMSLFDVHEIVEIGPFTIPQVDVPAIPLEIHESIHMDPIVLVPATTIPAQTRTIPLDIPASPGSTMTLPLISMRFEGEDWILGSTAAIPNFGDPFPAPTQGITIHTGPGPGTTGELKISIPGFEIPQIATTRFLLDVNISGGLPAFTLFAGGLTIPTNAIPLTIDASGALDPITIFPGGYTIDPLPLHLALNLTVPDSSIPIIDVPPTPGFGNTTATPSSGFFNSGAGGVSGFGNVGSNLSGWWNQAASALAGSGSGVLNVGTLGSGVLNVGSGVSGIYNTSVLPLGTPAVLSGLGNVGHQLSGVSAAGTALNQIPILNIGLADVGNFNVGFGNVGDVNLGAANLGAQNLGLGNVGTGNLGFANVGHGNIGFGNSGLTAGAAGLGNTGFGNAGSANYGFANQGVRNIGLANTGTGNIGIGLVGDNLTGIGGLNSGAGNIGLFNSGTGNIGFFNSGTGNFGIGNSGSFNTGIGNSGTGSTGLFNAGSFNTGVANAGSYNTGSFNAGDTNTGGFNPGTINTGWFNTGHTNTGIANSGNVGTGAFMSGNFSNGLLWRGDHEGLFSLFYSLDVPRITIVDAHLDGGFGPVVLPPIPVPAVNAHLTGNVAMGAFTIPQIDIPALTPNITGSAAFRIVVGSVRIPPVSVIVEQIINASVGAEMRIDPFEMWTQGTNGLGITFYSFGSADGSPYATGPLVFGAGTSDGSHLTISASSGAFTTPQLETGPITLGFQVPGSVNAITLFPGGLTFPATSLLNLDVTAGAGGVDEPAITWPEIAASADGSVYVLASSIPLINEPPTPGIGNSTITPSSGFFNAGAGGGSGFGNFGAGTSGWWNQAHTALAGAGSGFANVGTLHSGVLNLGSGVSGIYNTSTLGVGTPALVSGLGNVGHQLSGLLSGGSAVNPVTVLNIGLANVGSHNAGFGNVGEVNLGAANLGAHNLGFGNIGAGNLGFGNIGHGNVGVGNSGLTAGVPGLGNVGLGNAGGNNWGLANVGVGNIGLANTGTGNIGIGLTGDYQTGIGGLNSGAGNLGLFNSGAGNVGFFNTGTGNFGLFNSGSFNTGVGNSGTGSTGLFNAGSFNTGVANAGSYNTGSFNVGDTNTGGFNPGSINTGWLNAGNANTGVANAGNVNTGAFVTGNFSNGILWRGDYQGLAGFAVGYTLPLFPAVGADVSGGIGPITVLPPIHIPPIPVGFAAVGGIGPIAIPDISVPSIHLGLDPAVHVGSITVNPITVRTPPVLVSYSQGAVTSTSGPTSEIWVKPSFFPGIRIAPSSGGGATSTQGAYFVGPISIPSGTVTFPGFTIPLDPIDIGLPVSLTIPGFTIPGGTLIPTLPLGLALSNGIPPVDIPAIVLDRILLDLHADTTIGPINVPIAGFGGAPGFGNSTTLPSSGFFNTGAGGGSGFSNTGAGMSGLLNAMSDPLLGSASGFANFGTQLSGILNRGAGISGVYNTGALGVVTAAVVSGFGNVGQQ LSGLLFTGVGP(SEQ ID NO: 12, TUBERCULIST No. 3350c, asavailable Mar. 1, 2007, herein incorporatedby reference, also known as PPE56 or PPE.

In a second embodiment, an Mtb polypeptide of use in the methodsdisclosed herein has a sequence at least 75%, 85%, 90%, 95%, 96%, 97%,98% or 99% homologous to the amino acid sequence set forth in one of SEQID NOs: 1-12. For example, the polypeptide can have an amino acidsequence, at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to oneof the amino acid sequences set forth in SEQ ID NOs: 1-12. Exemplarysequences can be obtained using computer programs that are readilyavailable on the internet and the amino acid sequences set forth herein.In one example, the polypeptide retains a function of the Mtb protein,such as binding to an antibody that specifically binds the Mtb epitope.

Minor modifications of an Mtb polypeptide primary amino acid sequencesmay result in peptides which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein. Thus, a specific, non-limitingexample of a Mtb polypeptide is a conservative variant of the Mtbpolypeptide. A table of conservative substitutions is provided herein.Substitutions of the amino acids sequence shown in SEQ ID NOs: 1-12 canbe made based on this table.

Mtb polypeptides are disclosed herein that can be used to induce animmune response (immunogenic). These peptides include or consist of atleast nine amino acids, such as nine to twenty amino acids consecutiveamino acids of an Mtb polypeptide set forth above. Specific,non-limiting examples are twelve, eleven, ten amino acids, or nineconsecutive amino acids of one of the Mtb polypeptides set forth above.In these examples, the Mtb polypeptide does not include the full-lengthamino acid sequences set forth as SEQ ID NOs: 1-12.

An isolated polypeptide is disclosed that includes nine to twelveconsecutive amino acids from an Mtb polypeptide, wherein the isolatedpolypeptide comprises the amino acid sequence set forth as QTVEDEARRMW(SEQ ID NO: 13). In some embodiments, the polypeptide is nine, ten oreleven amino acids in length. In additional embodiments, the polypeptideconsists of the amino acid sequence set forth as SEQ ID NO: 13. Anisolated polypeptide is disclosed that includes nine to twelveconsecutive amino acids from an Mtb polypeptide, wherein the isolatedpolypeptide comprises the amino acid sequence set forth as VSAAIAGLF(SEQ ID NO: 14). In some embodiments, the polypeptide is nine, ten oreleven amino acids in length. In additional embodiments, the polypeptideconsists of the amino acid sequence set forth as SEQ ID NO: 14.

In several embodiments, the isolated Mtb polypeptide is included in afusion protein. Thus, the fusion protein can include the Mtb polypeptide(see above) and a second heterologous moiety, such as a myc protein, anenzyme or a carrier (such as a hepatitis carrier protein or bovine serumalbumin) covalently linked to the Mtb polypeptide. In several examples,a polypeptide consisting of nine to twelve amino acids of one of theamino acid sequences set forth as SEQ ID NOs: 1-14 that bind MHC class Iis covalently linked to a carrier. In additional example, a polypeptideconsisting of one of the amino acid sequences set forth as one of SEQ IDNOs: 1-14 is covalently linked to a carrier.

In additional examples, the polypeptide can be a fusion protein and canalso include heterologous sequences to Mtb (such as amino acid sequencesof at least nine amino acids in length that are not included in SEQ IDNO: 1). Thus, in several specific non-limiting examples, the immunogenicpeptide is a fusion polypeptide, for example the polypeptide includessix sequential histidine residues, a β-galactosidase amino acidsequence, or an immunoglobulin amino acid sequence. The polypeptide canalso be covalently linked to a carrier. Suitable carriers include, butare not limited to, a hepatitis B small envelope protein HBsAg. Thisprotein has the capacity to self assemble into aggregates and can formviral-like particles. The preparation of HBsAg is well known, see forexample European Patent Application Publication No. EP-A-0 226 846,European Patent Application Publication No. EP-A-0 299 108 and PCTPublication No. WO 01/117554, and the amino acid sequence disclosed, forexample, in Tiollais et al., Nature, 317: 489, 1985, and European PatentPublication No. EP-A-0 278 940, and PCT Publication No. WO 91/14703, allof which are incorporated herein by reference.

In additional embodiments, the protein consists of the Mtb polypeptide.A second heterologous moiety can be non-covalently linked to the Mtbpolypeptide. For example, a polypeptide consisting of nine to twelveconsecutive amino acids of one of the proteins set forth as one of SEQID NO: 1-14 can be non-covalently linked to a carrier.

The polypeptide can optionally include repetitions of one or more of theMtb polypeptides disclosed herein. In one specific, non-limitingexample, the polypeptide includes two, three, four, five, or up to tenrepetitions of one of the Mtb polypeptides described above.Alternatively, more than one polypeptide can be included in a fusionpolypeptide. Thus, in several examples, the polypeptide can include atleast two, at least three, at least four, at least five or at least sixof the amino acid sequences set forth as SEQ ID NOs: 1-14. A linkersequence can optionally be included between the Mtb polypeptides.

The Mtb polypeptides disclosed herein can be chemically synthesized bystandard methods, or can be produced recombinantly. An exemplary processfor polypeptide production is described in Lu et al., Federation ofEuropean Biochemical Societies Letters. 429:31-35, 1998. They can alsobe isolated by methods including preparative chromatography andimmunological separations.

If desired, polypeptides can also be chemically synthesized by emergingtechnologies. One such process is described in W. Lu et al., Federationof European Biochemical Societies Letters. 429:31-35, 1998. Polypeptidescan also be produced using molecular genetic techniques, such as byinserting a nucleic acid encoding Mtb or an epitope thereof into anexpression vector, introducing the expression vector into a host cell,and isolating the polypeptide (see below).

An Mtb polypeptide can be covalently linked to a carrier, which is animmunogenic macromolecule to which an antigenic molecule can be bound.Carriers are chosen to increase the immunogenicity of the bound moleculeand/or to elicit higher titers of antibodies against the carrier whichare diagnostically, analytically, and/or therapeutically beneficial.Covalent linking of a molecule to a carrier can confer enhancedimmunogenicity and T cell dependence (see Pozsgay et al., PNAS96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976; Dintzis etal., PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers,which can be natural (for example, polysaccharides, polypeptides orproteins from bacteria or viruses), semi-synthetic or syntheticmaterials containing one or more functional groups to which a reactantmoiety can be attached. Bacterial products and viral proteins (such ashepatitis B surface antigen and core antigen) can also be used ascarriers, as well as proteins from higher organisms such as keyholelimpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serumalbumins, and mammalian immunoglobulins. Additional bacterial productsfor use as carriers include bacterial wall proteins and other products(for example, streptococcal or staphylococcal cell walls andlipopolysaccharide (LPS)).

Polynucleotides encoding the Mtb polypeptides disclosed herein are alsoprovided. Exemplary nucleic acid sequences are set forth below:

ESXJ (ESAT-6 LIKE PROTEIN 2) (SEQ ID NO: 15)atggcctcgcgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatctcgggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgaacagcaagagcaggcctcccagcagatcctcagcagctgaESXK (ESAT-6 LIKE PROTEIN 3) (SEQ ID NO: 16)atggcctcacgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatttccggtgcgggctggagtggcatggccgaggcgacctcgctagacaccatggcccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgagcagcaagagcaggcctcccagcagatcctcagcagctaaESXM (ESAT-6 LIKE PROTEIN ESXM) (SEQ ID NO: 17)atggcctcacgttttatgacggatccgcatgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatttccggtgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacctagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgaacagcaagagcaggcctcccagcagatcctgagcagctagESXP (ESAT-6 LIKEK PROTEIN 7) (SEQ ID NO: 18)atggcaacacgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatctcgggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatggcccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgagcagcaagagcaggcctcccagcagatcctcagcagctaaESXW (ESAT-6 LIKE PROTEIN 10) (SEQ ID NO: 19)atgacctcgcgttttatgacggatccgcacgcgatgcgggacatggcgggccgttttgaggtgcacgcccagacggtggaggacgaggctcgccggatgtgggcgtccgcgcaaaacatttccggcgcgggctggagtggcatggccgaggcgacctcgctagacaccatgacccagatgaatcaggcgtttcgcaacatcgtgaacatgctgcacggggtgcgtgacgggctggttcgcgacgccaacaactacgaacagcaagagcaggcctcccagcagatcctcagcagctga PE9 (PE FAMILY PROTEIN)(SEQ ID NO: 20) atgtcatacatgattgccacaccagcggcgttgacggcggcggcaacggatatcgacgggattggctcggcggttagcgttgcgaacgccgcggcggtcgccgcgacaaccggagtgctggccgccggtggcgatgaagtgttggcggccatcgctaggctgttcaacgcaaacgccgaggaatatcacgccctcagcgcgcaggtggcggcgtttcaaaccctgtttgtgcgcaccttgactggggggtgcggagtctttcgccggcgccgaggccgccaatgcgtcacagctgcagagcatcgcgcggcaggtgcggggcgccgtcaacgccgtcgccggtcaggtgacgggcaatggcggctccggcaacagcggcacttcggctgcggcggccaacccgaattccgacaacacagcGagcatcgccgatag PE_PGRS42 (PE-PGRS FAMILY PROTEIN)(SEQ ID NO: 21) gtgtcgttggtgatcgcgacgccgcagctgctggcaactgcggctttggatttagcgagtattggttcgcaggtgagcgcggctaatgcggccgcggcgatgccgacgacggaagtggtggctgcggctgccgatgaagtgtcggcggcgattgcggggttgttcggggcccatgctcggcagtatcaggcgctcagcgtacaggtggcagcgtttcacgagcagtttgtgcaggcgttgactgcggccgcgggtcggtatgccagcactgaggccgctgttgagcggagtctgctgggtgcggtgaatgcgcccaccgaggcgcttttggggcgcccgttgatcggaaacggcgccgacgggacggcacccgggcagcctggcgcggccggcgggttgctgtttggcaacggtggcaacggcgcggctggcgggttcggtcaaaccggcggcagcggaggcgcggccgggttgatcggcaacggcggcaacggcggggccggtggtaccggcgcggccggcggtgccggtgggaacggggggtggttgtggggcaacggcggcaacggcggtgtcggcggcaccagcgtggccgcaggcatcgggggtgcgggcggtaacggcggcaacgccgggctgttcggccatggcggcgccggtggtaccggcggcgccggcctcgccggggcaaacggggtcaatcccacgcccggccccgcggccagcaccggggacagcccggcagatgtgtccggcatcggtgatcaaaccggcggcgacggcggcacgggcggccatggcactgccggcacgccgaccggtggcaccggcggcgacggtgccaccgcgacggcaggctcgggcaaggccaccggcggtgccggtggtgacggcggtaccgccgctgccggtggcggcggcggcaacggcggcgacggcggagtcgcgcagggcgacattgcgagcgcctttggcggtgatggtggcaacgggtccgacggtgtagccgccggcagtgggggtggtagcggcggcgccggaggcggcgctttcgtacacatcgccactgccacctctaccggtggtagcggcggtttcggtggtaacggggctgccagtgccgcctccggcgccgacggtggcgcagggggagctggcggcaatggtggcgccggcgggttgctattcggtgatggcggcaacggtggcgccggtggcgcgggtggtatcggtggtgacggcgccacgggggggcccgggggaagcggcggcaacgctggcatcgcgaggtttgacagcccagaccccgaggcagaacccgatgtggtcggcggcaagggtggtgatggcggcaagggcggcagcggccttggcgtcggcggcgccggcgggaccggcggcgcgggcggcaacggcggcgccggcgggttgttgttcggcaacggcggcaacggcggcaacgccggggccggcggggatggcggcgccggcgttgccggtggggttggcggtaacggcggcggtggtggcaccgcgacgtttcacgaagacccggtcgctggtgtctgggcggtcggtggcgtaggtggtgatggtggctccggcggcagctcgcttggtgtcggcggggtgggcggagccggtggcgtgggtggcaagggtggcgccagcggcatgttgatcggcaacggcggcaacggtggcagcggcggagtcggtggggccggtggagtcggcggggctggcggtgacggcggcaacggcggctccggtggcaacgccagtacttttggcgatgagaactccatcggcggggccggcgggacgggcggcaacgggggcaacggcgcaaacggcggtaacggtggcgctggcggtattgccggcggtgcgggtgggtccggagggttcctcagcggtgccgcaggagtcagcggcgctgacggtatcggtggcgcgggcggcgcaggcggtgccggtggcgcgggcggtagcggcggtgaggcaggcgcggggggcctcaccaacggccccgggtcccctggcgtttccggcaccgaaggcatggccggcgcgcccggctagRv1860 (FIBRONECTIN ATTACHMENT PROTEIN) (SEQ ID NO: 22)atgcatcaggtggaccccaacttgacacgtcgcaagggacgattggcggcactggctatcgcggcgatggccagcgccagcctggtgaccgttgcggtgcccgcgaccgccaacgccgatccggagccagcgcccccggtacccacaacggccgcctcgccgccgtcgaccgctgcagcgccacccgcaccggcgacacctgttgcccccccaccaccggccgccgccaacacgccgaatgcccagccgggcgatcccaacgcagcacctccgccggccgacccgaacgcaccgccgccacctgtcattgccccaaacgcaccccaacctgtccggatcgacaacccggttggaggattcagcttcgcgctgcctgctggctgggtggagtctgacgccgcccacttcgactacggttcagcactcctcagcaaaaccaccggggacccgccatttcccggacagccgccgccggtggccaatgacacccgtatcgtgctcggccggctagaccaaaagctttacgccagcgccgaagccaccgactccaaggccgcggcccggttgggctcggacatgggtgagttctatatgccctacccgggcacccggatcaaccaggaaaccgtctcgctcgacgccaacggggtgtctggaagcgcgtcgtattacgaagtcaagttcagcgatccgagtaagccgaacggccagatctggacgggcgtaatcggctcgcccgcggcgaacgcaccggacgccgggccccctcagcgctggtttgtggtatggctcgggaccgccaacaacccggtggacaagggcgcggccaaggcgctggccgaatcgatccggcctttggtcgccccgccgccggcgccggcaccggctcctgcagagcccgctccggcgccggcgccggccggggaagtcgctcctaccccgacgacaccgacaccgcagCggaccttaccggcctgaRv1273c (PROBABLE DRUGS-TRANSPORT TRNASMEMBRANEATP-BINDING PROTEIN ABC TRANSPROTER) (SEQ ID NO: 23)atgctcctggccctgctgcgccagcacatccgaccgtaccgccggctggtcgcgatgctgatgatgctgcagctggtcagcaccctggcttcgctatacctcccgacggtcaacgccgcaatcgtcgacgacggcgtcgccaagggcgacaccgccaccatcgtacggctgggtgcggtgatgcttggggtgaccggattgcaggtgctgtgcgcgatcggggcggtctatctgggctcccggaccggggcgggtttcggccgtgacctgcgctcggcaatgttcgaacacatcatcaccttctcggaacgcgagaccgcccgattcggcgctccgacgttgttgacccgcagcaccaacgacgtccggcagatcctgttcctggtccagatgaccgccaccgtgctggtcaccgcaccgatcatgtgcgtcggcggaatcatcatggccatccaccaggaggccgcgctgacatggctgctgctggtcagcgttccgattctggccgtagcaaactactggatcatctcccacatgctgccgctcttccgccgcatgcagagcctgatcgacggcatcaaccgggtgatgcgcgatcagctgtccggggtgcgagtggtccgcgccttcacccgcgaaggctatgaacgcgacaagttcgcgcaggccaatacggcgctgtcgaatgccgcactgagcgccggcaactggcaagcactgatgctgccggtgaccacgctgaccatcaacgcatccagcgtcgcactgatctggttcggtgggctacgcatcgacagcggccagatgcaggtcggctccctgatcgccttcctgtcctacttcgcccagatcctgatggcggtgttgatggcgaccatgacgctggccgtgctgccacgagcgtcggtctgcgccgaacgcatcaccgaggtgctttccacgcccgccgcactcggtaaccccgacaatcccaagttcccgacggacggggtcacgggcgtagtgcgcttggctggcgcaacctttacctatcctggcgccgactgcccggtgctgcaggacatttcgttgactgcgcggcccggtaccaccaccgcgatcgtcggcagtaccggttcgggcaagtcgacactggtgtcgttgatctgccggctctacgacgtcaccgctggcgcggtcttggttgacggtatcgacgtccgcgagtaccacaccgagcggctctggtcagcgatcgggctggtgccccagcgcagctacctcttctccggaaccgtcgcggacaacctgcgctacggcgggggcccagaccaggtagtcaccgagcaggagatgtgggaggcgctgcgggtcgccgcggccgacggctttgtacaaacagacgggctgcagacgcgtgtcgcccaaggtggtgtcaacttctccggcgggcagcgccaacggctggcgatagcccgagcggtcatccgacgtccggccatctatgtgttcgacgacgcgttctccgcacttgacgtgcacaccgacgccaaagtccacgcatcgctgcgacaggtatctggtgatgcaaccatcattgttgttacacaacggatttcgaatgccgctcaggccgaccaggtcatcgttgtcgataacggtaagatcgtcggcacgggcacccacgaaacgctgctggccgattgccccacctatgccgaattcgccgcctcacaatcgctgagcgccacggtcgggggtGtagggtgaRv0159c (PE FAMILY PROTEIN) (SEQ ID NO: 24)atgtcctacgtcatcgcggccccggagatgttggcaacgacggccgcggacgtggacgggatcggttcggcgatacgagcggccagcgcgtccgctgcgggtccaacgaccggactgctggccgcggccgccgatgaggtgtcgtcggccgctgcagcgctgttcagcgaatacgcgcgcgaatgtcaagaggtcctaaagcaggctgcggcgttccatggcgagttcacccgggcgctggctgccgccggggccgcctatgcccaggctgaagccagcaacaccgctgctatgtcgggcaccgccgggtccagcggcgccctcggttctgtcgggatgctgtcaggcaacccgctaaccgcgttgatgatgggcggcaccggggaaccgatccttagtgaccgcgtcttggcgatcattgacagcgcatacattcggcccattttcgggcccaacaacccggtcgcccagtacacgcccgagcagtggtggccgtttatcgggaacctgtcactggaccaatccatcgcccagggtgtcacgctgctgaacaacggcatcaacgcggaactacaaaatgggcatgacgtcgtcgttttcggctactcgcaaagcgccgcggtagcgaccaatgaaatacgcgctcttatggcgttaccaccgggccaagccccagatccaagccggctggctttcacgttgatcggtaatatcaataaccccaacggcggcgtcctcgagcgttacgtgggcctttacctcccgttcttggatatgtcgttcaacggtgcgactccaccggattccccctaccagacctacatgtacaccggccaatacgacggctacgcccacaacccgcagtacccgctcaatatcttgtcggacctcaacgccttcatgggcatcagatgggtgcacaacgcgtaccccttcaccgcggccgaggttgccaatgccgtgccgttgcccacgtctccgggctacaccggcaacacccattactacatgtttctgacccaggacctgccgctgttgcagccgattcgcgccatccccttcgtagggaccccaatagccgagctgattcagcccgacctacgggtgctagtcgacttgggctatggctacggctacgccgacgtacccaccccggccagcctgttcgcgccaatcaacccgatcgccgtggcctcggccctggcgaccgggaccgtgcaaggcccccaagccgccctagtaagcatcggattgttaccgcagtccgcgctacccaatacgtatccgtatcttccgtcggcgaatccgggcctgatgttcaacttcggtcaatccagtgtgacggagttgtcggtgctcagtggcgccctcgggtccgtagcgagattgattccaccgat cgcgtgaRv3350c (PPE FAMILY PROTEIN) (SEQ ID NO: 25)atggagtttccggtgttgccaccggaaatcaactccgtgctgatgtattcgggtgcggggtcgagcccgttgctggcggcggccgcggcgtgggatgggctggctgaggagttggggtcggcggcggtgtcgtttgggcaggtgacgtcgggcctgacggcgggggtgtggcagggtgcggcggcggcggcgatggcggccgcggcggcgccgtatgcggggtggttgggttcggtggcggccgcggccgaggcggtggccgggcaggcgcgggtggtggtgggggtctttgaggcggcgttggcggcgacggtggatccggcgctggtggcggccaaccgggcgcggctggtggcgttggcggtgtcgaatctgttggggcagaacacgccggcgatcgcggccgccgaggccgagtacgagctgatgtgggccgccgatgtggcggcgatggccggctaccattccggcgcgtcggctgctgccgcggcgttgccggcgttcagcccaccggcgcaggcgctggggggaggtgtcggcgcgttccttaccgccctgttcgccagccctgcgaaggcgctgagcctgaatgcgggtttgggcaatgtcggcaattacaacgtcgggttgggcaatgtcggggtgttcaacctgggcgcgggcaatgtgggtgggcagaatctgggtttcgggaatgccggtggcaccaatgtcgggttcggcaacctcggtaacgggaatgtcgggttcggcaactccggtctgggggcgggcctggccggcttgggcaatatcgggttgggcaatgcgggcagcagcaactatggtttcgcaaacctgggtgtgggcaacatcggtttcggcaacaccggcaccaacaacgtcggcgtcgggctcaccggcaaccacctgacgggtatcgggggcctgaattcgggcaccgggaatatcgggttgttcaactccggcaccgggaatgtggggttcttcaattcggggaccgggaacttcggggtgttcaactcgggtaattacaacaccggtgtcggtaatgcggggacggccagcacggggttgttcaatgccggcaatttcaacaccggcgtggtgaacgtgggcagttacaacaccggcagtttcaacgccggcgacaccaacaccggtggcttcaaccccggcggtgtgaacaccggctggctgaacaccggcaacaccaacaccggcatcgccaactcgggcaacgtcaacaccggcgcgttcatctcgggcaacttcaacaacggcgtgctgtgggtgggtgactaccagggcctgttcggcgtctccgccggctcgtcgatccccgcaattcccatcggcctggtgctcaacggcgacatcggcccgatcaccatccagcccatcccgatcctgcccaccatcccgctcagcattcaccaaaccgtcaacttgggcccgctggtggttcccgacatcgtgatccccgccttcggcggcggtatcggcatacccatcaacatcggcccgctgaccatcacacccatcaccctgtttgcccaacagacatttgtcaaccaattgccctttcccaccttcagtttagggaaaatcacaattccacaaatccaaacctttgattctaacggtcagcttgtcagctttatcggccctatcgttatcgacaccaccattcccggacccaccaatccacagattgatttaacgatcagatgggatacccctccgatcacgctgttcccgaatggcatcagtgctcccgataatcctttggggttgctggtgagtgtgtcgatcagtaacccgggctttaccatcccgggatttagtgttcccgcgcagccgttgccgttgtcgatcgatatcgagggccagatcgacgggttcagcaccccgccgatcacgatcgatcgcatccccctgaccgtggggggcggggtcacgatcggccccatcacgatccagggccttcatatcccggcggcgccgggagtggggaacaccaccacggccccgtcgtcgggattcttcaactccggtgcgggtggggtgtcgggtttcggcaacgtcggcgcgggcagctcgggctggtggaaccaggcgccgagcgcgctgttgggggccggttcgggtgttggcaacgtgggcaccctgggctcgggtgtgctcaacctgggctcagggatctcggggttctacaacaccagcgtgttgcctttcgggacaccggcggcggtgtcgggcatcggcaacctgggccagcagctgtcgggggtgtcggcggcgggaaccacgctgcgctcgatgctcgccggcaacctcgggttggccaatgtgggcaacttcaacaccgggttcggaaatgtcggggacgtcaacctgggtgcggccaacatcggtgggcacaacctgggcctgggcaatgtcggggacggcaacctggggttgggcaacatcggccatggcaacctggggtttgccaacttgggcctgaccgccggcgcggcgggggtgggcaatgttggttttggcaatgccggcatcaacaactatggcttggcgaacatgggtgtgggcaatattgggtttgccaacaccggcacgggcaacatcgggatcgggctggtcggggaccatcggaccgggatcgggggcttgaactccggcatcggcaatatcgggttgttcaactccggcaccggcaacgtcgggttcttcaattccgggaccggcaacttcggcatcgggaactccggccgcttcaacaccgggatcggtaatagcggaacggccagcaccgggctcttcaatgccggcagcttcagcaccggcatcgccaacactggtgactacaacacgggcagcttcaacgccggcgacaccaacaccggtggcttcaacccgggcggcatcaacaccggctggttcaacaccgggcatgccaacaccgggttggccaacgcgggcaccttcggcaccggcgccttcatgacgggcgactacagcaacggcctgttgtggcggggcggctacgagggcctggtcggcgtccgcgtcgggcccacgatctcccaattcccggtcaccgtgcacgcgatcggcggggtgggcccgctgcatgtggcgcccgtcccggtacccgccgtgcacgtcgagatcaccgacgccaccgtcggcctgggtccgttcaccgtcccaccgatcagcattccctcacttcccatcgccagcatcaccggaagcgtggacctggccgcaaacaccatctcgccgattcgcgctcttgacccgctcgccggttcgatagggctttttctcgagccgttccgcctcagtgacccatttatcaccattgatgcgttccaagttgttgccggtgtcttgttcctagagaacatcattgtgcccggcctcacggttagcggtcagatattggtcaccccgacaccaattcccctaaccctcaacttggacaccaccccgtggacgcttttcccgaatggtttcaccattcccgcgcaaacccccgtgacggtgggtatggaggtcgccaacgacgggttcaccttcttcccgggtgggctgacctttccgcgggcctccgccggggtcaccggactgtccgtggggctggacgcgttcacgctgttgcccgacgggttcaccctcgacaccgtgccggcgaccttcgacggcaccatcctcatcggcgatatcccgatcccgatcatcgatgtgccggcggtgccggggttcggcaacaccaccacggccccatcgtcggggttcttcaacaccggcggcggcggtggatcggggttcgccaacgtcggcgcgggcacgtcgggctggtggaaccaggggcacgacgtgttagcaggggcgggctcgggagttgccaatgccggcacgctgagctcgggcgtgctgaacgtcggctcggggatctccgggtggtacaacaccagcaccctgggagcgggcaccccggcggtggtctcgggcatcggcaacctcggccagcagctgtcggggttcttggcaaatgggaccgtgctcaaccggagccccattgtcaatatcgggtgggccgatgtgggcgcgttcaacaccgggttgggcaatgtgggggacctcaactggggtgcggccaacatcggcgcgcagaacctgggcctgggcaatctcggcagcgggaacgtcgggttcggcaacatcggtgccggcaacgtcgggttcgccaactcgggtccggcggtgggcctggccggcctgggcaacgtggggttgagcaatgccggcagcaacaactgggggctggccaacctgggtgtgggcaacatcgggttggccaacaccggcacgggcaacatcgggatcgggctggtcggcgactaccagaccggcatcggcggcctcaactcgggtagtggcaatatcggattgttcaattccggcaccggcaatgtcgggttcttcaacaccggcaccggcaacttcggactgttcaactccggtagtttcaacaccggcatcggtaatagcggaaccggcagtactgggctcttcaatgccggcaatttcaacaccggcatcgccaaccccgggtcgtacaacacgggcagcttcaatgtcggtgataccaacaccggtggtttcaacccgggcgacatcaacaccggctggttcaacaccggcattatgaatacgggcacccgcaacaccggcgccctcatgtcggggaccgacagcaacggcatgctgtggcgcggcgaccacgagggcctgttcggcctgtcctatggcatcacgatcccgcaattcccgatccgcatcaccacgactggcggtatcggccccatcgtcatcccggacaccacgatccttccgccgctgcacctgcagatcaccggcgacgcggactacagcttcaccgtgcccgacatccccatccccgccatccacatcggcatcaatggcgtcgtcaccgtcggcttcaccgccccggaagccaccctgctgtccgccctgaagaataacggtagcttcatcagcttcggccccatcacgctctcgaatatcgatattccgcccatggatttcacgttaggcctgcccgttcttggtcctatcacgggccaactcggaccaattcatcttgagccaatcgtggtggccgggatcggtgtgcccctggagatcgagcccatccccctggatgcgatttcgttgagtgagtcgattcctatccgcatacctgttgatattccggcctcggtcatcgatgggatttcaatgtcggaagtggtgccgatcgatgcgtccgtggacatcccggcggtcacgatcacaggcaccaccatttccgcgatcccgctgggcttcgacattcgcaccagtgccggacccctcaacatcccgatcatcgacatcccggcggcgccgggcttcgggaactcgacccagatgccgtcgtcggggttcttcaacaccggtgccggcggcggatcgggcatcggcaacttgggtgcgggcgtgtcgggcctgctcaaccaggccggcgcggggtcactggtggggacactctcggggctgggcaatgccggcaccctggcctcgggtgtgctgaactccggcaccgccatctccgggctgttcaacgtgagcacgctggacgccaccaccccggcggtgatctcggggttcagcaacctcggcgaccatatgtcgggggtgtccatcgatggcctgatcgcgatcctcaccttcccacctgccgagtccgtgttcgatcagatcatcgacgcggccatcgccgagctgcagcacctcgacatcggcaacgctttggccttgggcaatgtcggcggggtgaacctcggtttggctaacgtcggtgagttcaacctgggtgcgggcaacgtcggcaacatcaacgtcggcgccggcaacctcggcggcagcaacttggggttgggcaacgtcgggaccggcaacctcgggttcggcaacatcggtgccggcaatttcggattcggcaacgcgggcctgaccgcgggcgcggggggcctgggcaatgtggggttgggtaacgccggcagcggcagctgggggttggccaacgtgggtgtgggcaatatcgggttggccaacaccggcaccggcaacatcgggatcgggctgaccggggactatcggaccgggatcggcggcctgaactcgggcaccgggaacctcgggttgttcaactcgggcaccggcaacatcgggttcttcaacaccgggaccgggaacttcgggctgttcaactcgggcagttacagcaccggtgtggggaatgcgggcacggccagcaccgggttgttcaacgcggggaacttcaacaccggtctggccaatgccggctcctacaacaccggcagcctcaacgtgggcagcttcaacaccggcggcgtcaacccgggcaccgtcaacaccggctggttcaacaccggccacaccaacaccggcctgttcaacaccggcaacgtcaacaccggcgcgttcaactccggcagcttcaacaacggggcgctgtggaccggtgactaccacgggctggtcggcttctccttcagcatcgacatcgccggcagcaccctgctggacctcaacgaaaccctcaacctgggccccatccacatcgagcagatcgacatccccggcatgtcgctgttcgacgtccacgaaatcgtcgagatcggacccttcaccatcccgcaggtcgatgttcccgcgataccgctagagatccacgaatcgatccacatggatcccatcgtcctggtgcccgccaccacaattcccgcacagacgagaaccattccgctggacatccccgcctcacccgggtcaaccatgacgcttccgctcatcagcatgcgcttcgaaggcgaggactggatcctcgggtcgaccgcggcgattcccaatttcggagaccccttcccggcgcccacccagggcatcaccattcacaccggccctggccccggaacgaccggcgagctcaagatatctattccgggtttcgagattccgcaaatcgctaccacgagattcctgttggacgtgaacatcagcggtggtctgccggccttcaccttgttcgcgggtggcctgacgatccccacgaacgccatcccgttaacgatcgatgcgtccggcgcgctggatccgatcacgattttcccgggtgggtacacgatcgacccgctgccgctgcacctggcgctgaatctcaccgtgcccgacagcagcatcccgatcatcgatgtcccgccgacgccagggttcggcaacaccacggcgaccccgtcgtcggggttcttcaactccggcgccggtggggtgtcggggttcggaaacgtcgggtcgaacctgtcgggctggtggaaccaggcggcgagcgcgctggcggggtcgggatcgggggtgttgaatgtcggcacgctgggctcgggtgtgctcaacgtcggctcgggtgtctcggggatctacaacaccagcgtgttgccgctcgggacgccggcggtgctgtcgggcctcggcaacgtcggccatcagctgtcgggcgtgtctgcggccgggaccgcgttgaaccagatccccatcctcaacatcgggttggcggatgtgggcaacttcaacgtcgggttcggcaacgtcggggacgttaacctgggcgcggccaacctcggtgcgcaaaacctggggctgggcaacgtcggcaccggcaacctcggcttcgccaacgtcggccacggcaatatcggtttcggcaattcgggtctgaccgccggcgcggccggcctgggcaacacggggttcggcaatgccggcagcgccaactatggtttcgccaaccagggcgtgcgcaacatcgggttggccaacaccggcaccggcaacatcgggatcgggctggtgggggacaacctcaccggcatcgggggcctgaactccggtgccggcaatatcggcttgttcaactccggcaccggcaacatcgggttcttcaactccgggaccggcaacttcggcatcggtaactcgggcagcttcaacaccggcatcggcaatagcggaacgggcagcactgggctcttcaatgccggcagcttcaacaccggcgtggccaacgccggcagctacaacaccggcagcttcaatgccggcgacaccaacaccggggggttcaacccgggcaccatcaacaccggctggttcaacaccggccacaccaataccggcatcgccaactcgggcaacgtcggcaccggcgcgttcatgtcgggcaacttcagcaacggcctgttgtggcggggtgatcacgagggcctgttcagcctgttctacagcctcgacgtgccccggatcaccatcgtggacgcccacctcgacggcggcttcggacccgtggtcctcccgcccatcccggtgccggccgttaatgcgcacctgaccggaaacgtcgcgatgggcgcattcaccattccgcagatcgacatccccgcactcaccccaaacatcaccggaagcgccgccttccgcatcgttgtggggtccgtgcgcattccgccggtgagtgtcattgtggagcaaataatcaacgcctcggttggggcggagatgaggatagatcccttcgaaatgtggactcaaggcactaatggccttggtataaccttctattcattcggatcggccgacggttcgccctacgccaccggcccactcgttttcggcgccggcacgagcgacggaagccatctcaccatttccgcgtccagcggggcgtttaccactccgcagctcgaaactggcccgatcacgttgggcttccaggtgcccggcagcgtcaacgcgatcaccctcttccccggtggtttgacgttcccggcgacctcgctgctgaacctggacgtgaccgccggcgccggcggcgtggacatcccggccatcacctggcccgagatcgcggcgagcgccgacggctcggtgtatgtcctcgccagcagcatcccgctgatcaacatcccgcccaccccgggcattgggaacagcaccatcaccccgtcgtcgggcttcttcaacgccggcgcgggcgggggatcgggcttcggcaacttcggcgcgggcacctcgggctggtggaaccaggcgcacaccgcgctggcgggggcgggctcgggttttgccaacgttggcacgctgcattccggtgtgctcaacctgggctcgggtgtctcggggatctacaacaccagcacgctgggggtggggaccccggcgctggtctcaggcctgggcaacgtcggccaccaactgtcggggctgctttccggcgggtccgcggtgaacccggtgaccgttctgaatatcgggttggccaacgtcggcagccacaacgccggtttcggcaatgtcggggaggtcaacctgggcgcggccaacctcggcgcgcacaacctgggcttcggaaatatcggcgccggcaacctggggttcggcaatattggccacggcaatgtcggagtcggcaactcgggtctgaccgcgggcgtgccgggcctgggcaatgtggggttgggcaatgccggcggcaacaactgggggttggccaacgtgggcgtgggcaatatcgggttggccaacaccggcaccggcaacattgggatcgggctgaccggcgactaccagaccggcatcggcggcctaaattccggtgccggcaacctggggttgttcaactccggcgccggcaacgtcgggttcttcaacaccgggaccggcaacttcgggttgttcaactccggcagcttcaacaccggcgtcggcaatagcggaacgggcagcactgggctcttcaatgccggcagtttcaacaccggtgtggccaacgccggcagctacaacacgggcagcttcaatgtcggtgacaccaacaccgggggcttcaacccgggcagcatcaacaccggctggctcaacgccggcaacgccaacaccggggtggccaacgcgggcaatgtcaacaccggcgccttcgtcaccggcaacttcagcaacggcatcctgtggcgcggcgactaccagggcctggccggcttcgccgtgggctacaccctcccgctgttccccgcggtgggcgccgacgtcagcggcgggatcggcccgattaccgtgctgccgcccatccacatcccgcccattccggtcggcttcgccgcggtcggtggcatcggcccgatcgccatcccggacatctctgttccatccattcacttgggcctcgaccccgccgtccatgtcggctccatcaccgtcaaccccattaccgtcaggaccccgcccgtgctcgtcagttactcccaaggagccgtcaccagcacgtccggaccaacctcagagatttgggtcaagcccagcttcttccccggaatccggatcgcgccctctagcggcgggggtgcaacgtccacgcaaggggcatactttgtggggcccatctccatcccctccggcacggtgaccttcccgggattcaccatccccctcgacccgatcgacatcggcctgccggtgtcgctgaccatcccggggttcaccatcccgggcggcaccctgatccccaccctcccgctgggcctcgcgttgtccaatggcatcccgcccgtcgacatcccggccatcgttctcgaccggatcttgctggacctgcacgccgacaccactatcggcccgatcaacgtcccgatcgccgggttcggcggggcgccgggtttcgggaactcgaccacgctgccgtcgtcgggcttcttcaacaccggagctggcggcggttcgggctttagcaacaccggcgcgggcatgtcgggattgctcaacgcgatgtcggatccgctgctcgggtcggcgtcgggcttcgccaacttcggcacccagctctecggcatcctcaaccgcggcgccggcatctcgggcgtgtacaacaccggcgcgctgggtgttgtcaccgcggccgtcgtctcgggtttcggcaacgtcggccagcaactgtcgggcttgctcttcaccggcgtcgggcccta a

These polynucleotides include DNA, cDNA and RNA sequences which encodethe polypeptide of interest. Silent mutations in the coding sequenceresult from the degeneracy (i.e., redundancy) of the genetic code,whereby more than one codon can encode the same amino acid residue.Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, orTTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC;asparagine can be encoded by AAT or AAC; aspartic acid can be encoded byGAT or GAC; cysteine can be encoded by TGT or TGC; alanine can beencoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA orCAG; tyrosine can be encoded by TAT or TAC; and isoleucine can beencoded by ATT, ATC, or ATA. Tables showing the standard genetic codecan be found in various sources (e.g., L. Stryer, 1988, Biochemistry,3.sup.rd Edition, W.H. 5 Freeman and Co., NY).

A nucleic acid encoding a Mtb polypeptide can be cloned or amplified byin vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the protein can be isolated by polymerase chainreaction of cDNA using primers based on the DNA sequence of themolecule. A wide variety of cloning and in vitro amplificationmethodologies are well known to persons skilled in the art. PCR methodsare described in, for example, U.S. Pat. No. 4,683,195; Mullis et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCRTechnology, (Stockton Press, NY, 1989). Polynucleotides also can beisolated by screening genomic or cDNA libraries with probes selectedfrom the sequences of the desired polynucleotide under stringenthybridization conditions.

The polynucleotides encoding a Mtb polypeptide include a recombinant DNAwhich is incorporated into a vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (such as a cDNA) independent ofother sequences. The nucleotides of the invention can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, may inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (20 or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (AMP) for propagation inbacteria. Plasmids for expression on K. lactis are known, such aspKLAC1. Thus, in one example, after amplification in bacteria, plasmidscan be introduced into the corresponding yeast auxotrophs by methodssimilar to bacterial transformation.

The Mtb polypeptides can be expressed in a variety of yeast strains. Forexample, seven pleiotropic drug-resistant transporters, YOR1, SNQ2,PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activatingtranscription factors, PDR1 and PDR3, have been simultaneously deletedin yeast host cells, rendering the resultant strain sensitive to drugs.Yeast strains with altered lipid composition of the plasma membrane,such as the erg6 mutant defective in ergosterol biosynthesis, can alsobe utilized. Proteins that are highly sensitive to proteolysis can beexpressed in a yeast lacking the master vacuolar endopeptidase Pep4,which controls the activation of other vacuolar hydrolases. Heterologousexpression in strains carrying temperature-sensitive (ts) alleles ofgenes can be employed if the corresponding null mutant is inviable.

Viral vectors can also be prepared encoding the Mtb polypeptidesdisclosed herein. A number of viral vectors have been constructed,including polyoma, SV40 (Madzak et al., 1992, J. Gen. Virol.,73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol.,158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia etal., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad.Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian(Brandyopadhyay et al., 1984, Mol. Cell. Biol., 4:749-754; Petropouploset al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top.Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell. Biol.,5:431-437; Sorge et al., 1984, Mol. Cell. Biol., 4:1730-1737; Mann etal., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990,J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol.,66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.).

Thus, in one embodiment, the polynucleotide encoding an Mtb polypeptideis included in a viral vector. Suitable vectors include retrovirusvectors, orthopox vectors, avipox vectors, fowlpox vectors, capripoxvectors, suipox vectors, adenoviral vectors, herpes virus vectors, alphavirus vectors, baculovirus vectors, Sindbis virus vectors, vacciniavirus vectors and poliovirus vectors. Specific exemplary vectors arepoxvirus vectors such as vaccinia virus, fowlpox virus and a highlyattenuated vaccinia virus (MVA), adenovirus, baculovirus and the like.

Pox viruses useful in practicing the present methods include orthopox,suipox, avipox, and capripox virus. Orthopox include vaccinia,ectromelia, and raccoon pox. One example of an orthopox of use isvaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripoxinclude goatpox and sheeppox. In one example, the suipox is swinepox.Examples of pox viral vectors for expression as described for example,in U.S. Pat. No. 6,165,460, which is incorporated herein by reference.Other viral vectors that can be used include other DNA viruses such asherpes virus and adenoviruses, and RNA viruses such as retroviruses andpolio.

In some cases, vaccinia viral vectors may elicit a strong antibodyresponse. Thus, while numerous boosts with vaccinia vectors arepossible, its repeated use may not be useful in certain instances.However, this sensitivity problem can be minimized by using pox fromdifferent genera for boosts. In one example, when the first or initialpox virus vector is vaccinia, the second and subsequent pox virusvectors are selected from the pox viruses from a different genus such assuipox, avipox, capripox or an orthopox immunogenically distinct fromvaccinia.

The vaccinia virus genome is known in the art. It is composed of a HINDF13L region, TK region, and an HA region. Recombinant vaccinia virus hasbeen used to incorporate an exogenous gene for expression of theexogenous gene product (see, for example, Perkus et al. Science229:981-984, 1985; Kaufman et al. Int. J. Cancer 48:900-907, 1991; MossScience 252:1662, 1991). A gene encoding an antigen of interest, such asan immunogenic Mtb polypeptide, can be incorporated into the HIND F13Lregion or alternatively incorporated into the TK region of recombinantvaccinia virus vector (or other nonessential regions of the vacciniavirus genome). Baxby and Paoletti (Vaccine 10:8-9, 1992) disclose theconstruction and use as a vector, of the non-replicating poxvirus,including canarypox virus, fowlpox virus and other avian species. Sutterand Moss (Proc. Nat'l. Acad. Sci. U.S.A. 89:10847-10851, 1992) andSutter et al. (Virology 1994) disclose the construction and use as avector, the non-replicating recombinant Ankara virus (MVA, modifiedvaccinia Ankara) in the construction and use of a vector.

Suitable vectors are disclosed, for example, in U.S. Pat. No. 6,998,252,which is incorporated herein by reference. In one example, a recombinantpoxvirus, such as a recombinant vaccinia virus is synthetically modifiedby insertion of a chimeric gene containing vaccinia regulatory sequencesor DNA sequences functionally equivalent thereto flanking DNA sequenceswhich in nature are not contiguous with the flanking vaccinia regulatoryDNA sequences that encode a Mtb polypeptide. The recombinant viruscontaining such a chimeric gene is effective at expressing the Mtbpolypeptide. In one example, the vaccine viral vector comprises (A) asegment comprised of (i) a first DNA sequence encoding a Mtb polypeptideand (ii) a poxvirus promoter, wherein the poxvirus promoter is adjacentto and exerts transcriptional control over the DNA sequence encoding anMtb polypeptide; and, flanking said segment, (B) DNA from a nonessentialregion of a poxvirus genome. The viral vector can encode a selectablemarker. In one example, the poxvirus includes, for example, a thymidinekinase gene (see U.S. Pat. No. 6,998,252, which is incorporated hereinby reference).

Viral vectors, such as poxviral vectors, that encode an Mtb polypeptideinclude at least one expression control element operationally linked tothe nucleic acid sequence encoding the Mtb polypeptide. The expressioncontrol elements are inserted in the viral vector to control andregulate the expression of the nucleic acid sequence. Examples ofexpression control elements of use in these vectors includes, but is notlimited to, lac system, operator and promoter regions of phage lambda,yeast promoters and promoters derived from polyoma, adenovirus,retrovirus or SV40. Additional operational elements include, but are notlimited to, leader sequence, termination codons, polyadenylation signalsand any other sequences necessary for the appropriate transcription andsubsequent translation of the nucleic acid sequence encoding the Mtbpolypeptide in the host system. The expression vector can containadditional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the one or more Mtb polypeptides areknown in the art. Such techniques involve, for example, homologousrecombination between the viral DNA sequences flanking the DNA sequencein a donor plasmid and homologous sequences present in the parentalvirus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419).In particular, recombinant viral vectors such as a poxyviral vector canbe used in delivering the gene. The vector can be constructed forexample by steps known in the art, such as steps analogous to themethods for creating synthetic recombinants of the fowlpox virusdescribed in U.S. Pat. No. 5,093,258, incorporated herein by reference.Other techniques include using a unique restriction endonuclease sitethat is naturally present or artificially inserted in the parental viralvector to insert the heterologous DNA.

Generally, a DNA donor vector contains the following elements: (i) aprokaryotic origin of replication, so that the vector may be amplifiedin a prokaryotic host; (ii) a gene encoding a marker which allowsselection of prokaryotic host cells that contain the vector (e.g., agene encoding antibiotic resistance); (iii) at least one DNA sequenceencoding the one or more Mtb polypeptide located adjacent to atranscriptional promoter capable of directing the expression of thesequence; and (iv) DNA sequences homologous to the region of the parentvirus genome where the foreign gene(s) will be inserted, flanking theconstruct of element (iii). Methods for constructing donor plasmids forthe introduction of multiple foreign genes into pox virus are describedin PCT Publication No. WO 91/19803, incorporated herein by reference.

Generally, DNA fragments for construction of the donor vector, includingfragments containing transcriptional promoters and fragments containingsequences homologous to the region of the parent virus genome into whichforeign DNA sequences are to be inserted, can be obtained from genomicDNA or cloned DNA fragments. The donor plasmids can be mono, di-, ormultivalent (i.e., can contain one or more inserted foreign DNAsequences). The donor vector can contain an additional gene that encodesa marker that will allow identification of recombinant virusescontaining inserted foreign DNA. Several types of marker genes can beused to permit the identification and isolation of recombinant viruses.These include genes that encode antibiotic or chemical resistance (e.g.,see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988,J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), aswell as genes such as the E. coli lacZ gene, that permit identificationof recombinant viral plaques by colorimetric assay (Panicali et al.,1986, Gene 47:193-199).

The DNA gene sequence to be inserted into the virus can be placed into adonor plasmid, such as an E. coli or a Salmonella plasmid construct,into which DNA homologous to a section of DNA such as that of theinsertion site of the poxvirus where the DNA is to be inserted has beeninserted. Separately the DNA gene sequence to be inserted is ligated toa promoter. The promoter-gene linkage is positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of pox DNA that isthe desired insertion region. With a parental pox viral vector, a poxpromoter is used. The resulting plasmid construct is then amplified bygrowth within E. coli bacteria and isolated. Next, the isolated plasmidcontaining the DNA gene sequence to be inserted is transfected into acell culture, for example chick embryo fibroblasts, along with theparental virus, for example poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively results in arecombinant poxvirus modified by the presence of the promoter-geneconstruct in its genome, at a site that does not affect virus viability.

As noted above, the DNA sequence is inserted into a region (insertionregion) in the virus that does not affect virus viability of theresultant recombinant virus. One of skill in the art can readilyidentify such regions in a virus by, for example, randomly testingsegments of virus DNA for regions that allow recombinant formationwithout seriously affecting virus viability of the recombinant. Oneregion that can readily be used and is present in many viruses is thethymidine kinase (TK) gene. The TK gene has been found in all pox virusgenomes examined, including leporipoxvirus (Upton et al., 1986, J.Virology 60:920); shope fibromavirus; capripoxvirus (Gershon et al.,1989, J. Gen. Virol. 70:525) Kenya sheep-1; orthopoxvirus (Weir et al.,1983, J. Virol. 46:530) vaccinia (Esposito et al., 1984, Virology135:561); monkeypox and variola virus (Hruby et al., 1983, PNAS 80:3411)vaccinia (Kilpatrick et al., 1985, Virology 143:399); Yaba monkey tumorvirus; avipoxvirus (Binns et al., 1988, J. Gen. Virol. 69:1275);fowipox; (Boyle et al., 1987, Virology 156:355); fowlpox (Schnitzlein etal., 1988, J. Virological Methods 20:341); fowlpox, quailpox; entomopox(Lytvyn et al., 1992, J. Gen. Virol. 73:3235-3240). In vaccinia, inaddition to the TK region, other insertion regions include, for example,the Hind111 M fragment. In fowlpox, in addition to the TK region, otherinsertion regions include, for example, the BamHI J fragment (Jenkins etal., 1991, AIDS Research and Human Retroviruses 7:991-998) theEcoRI-Hind111 fragment, EcoRV-HindIII fragment, BamHI fragment and theHind111 fragment set forth in EPO Application No. 0 308220 A1 (see alsoCalvert et al., 1993, J. Virol. 67:3069-3076; Taylor et al., 1988,Vaccine 6:497-503; Spehner et al., 1990; Boursnell et al., 1990, J. Gen.Virol. 71:621-628).

In swinepox, insertion sites include the thymidine kinase gene region.In addition to the requirement that the gene be inserted into aninsertion region, successful expression of the inserted gene by themodified poxvirus requires the presence of a promoter operably linked tothe desired gene. Generally, the promoter is placed so that it islocated upstream from the gene to be expressed. Promoters are well knownin the art and can readily be selected depending on the host and thecell type you wish to target. In one example, in poxviruses, pox viralpromoters are used, such as the vaccinia 7.5K, 40K or fowlpox promoterssuch as FPV C1A. Enhancer elements can also be used in combination toincrease the level of expression. Furthermore, inducible promoters canbe utilized.

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell can result in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo fibroblasts, HuTK143(human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infectionof cells with pox virus and transfection of these cells with plasmidvectors is accomplished by techniques standard in the art (see U.S. Pat.No. 4,603,112 and PCT Publication No. WO 89/03429).

Following in vivo recombination, recombinant viral progeny can beidentified by one of several techniques. For example, if the DNA donorvector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK- and can be selected on this basis (Mackett et al., 1982, Proc. Natl.Acad. Sci. USA 79:7415). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One specific non-limiting example of an indicator gene is theE. coli lacZ gene. Recombinant viruses expressing beta-galactosidase canbe selected using a chromogenic substrate for the enzyme (Panicali etal., 1986, Gene 47:193). Once a recombinant virus has been identified, avariety of well-known methods can be used to assay the expression of theMtb sequence encoded by the inserted DNA fragment. These methods includeblack plaque assay (an in situ enzyme immunoassay performed on viralplaques), Western blot analysis, radioimmunoprecipitation (RIPA), andenzyme immunoassay (EIA).

This disclosure encompasses a recombinant virus comprising more than oneantigen of interest for the purpose of having a multivalent vaccine. Forexample, the recombinant virus may comprise the virus genome or portionsthereof, the nucleic acid sequence encoding the Mtb polypeptide and anucleic acid sequence encoding a hepatitis B surface antigen or anyother carrier protein.

In one embodiment, a composition is provided that includes a recombinantvirus comprising a vaccinia virus genome or portions thereof, thenucleic acid sequence encoding an Mtb polypeptide and a recombinantvirus comprising the nucleic acid sequence encoding theimmunostimulatory molecule, B 7.1 alone or in combination with thenucleic acid sequence encoding the immunostimulatory molecule, B7-2, ora recombinant virus containing both the genes for an Mtb polypeptide andan immunostimulatory molecule. This disclosure also encompasses arecombinant virus comprising the Mtb polypeptide that is administeredwith a second recombinant virus comprising the virus genome or portionthereof, and one or more nucleic acid sequences encoding one or more B7molecules, such as a recombinant vaccinia virus expressing B7-1 and/orB7-2.

Thus, in one example, recombinant virus is disclosed that is arecombinant vaccinia virus containing B7-1 and a recombinant vacciniavirus containing B7-2 (designated rV-B7-1 and rV-B7-2, respectively);the composition can include rV-B7-1 and/or rV-B7-2 in combination withan immunogenic Mtb polypeptide.

The B7 molecule includes but is not limited to B7-1, B7-2 and analogsthereof. The B7 gene may be cloned from mammalian sources, including butnot limited to mammalian tissues, genomic libraries or cDNA libraries,such as from murine or human sources. Without being bound by theory,co-stimulatory molecules of the B7 family (namely B7-1, B7-2, andpossibly B7.3) are believed to be members of the immunoglobulin genesuperfamily. These molecules are present on macrophages, dendriticcells, monocytes (antigen presenting cells (APCs)). Significantamplification of the immune response against a given antigen generallydoes not occur without co-stimulation (June et al. (Immunology Today15:321-331, 1994); Chen et al. (Immunology Today 14:483-486); Townsendet al. (Science 259:368-370)). Freeman et al. (J. Immunol.143:2714-2722, 1989) report cloning and sequencing of B7-1 gene. Azumaet al. (Nature 366:76-79, 1993) report cloning and sequencing B7-2 gene.Thus, in one embodiment the B7-1 gene or the B7-2 genes are administeredin conjunction with the Mtb polypeptide. The insertion of nucleic acidsencoding B7-1 and B7-2 into vaccinia virus has been disclosed (see forexample, U.S. Pat. No. 6,893,869, incorporated herein by reference; thisU.S. patent also discloses the use of a nucleic acid encoding IL-2 in avaccinia virus). Several vectors including IL-2, B7-1 and B7-2 have beendeposited with the American Type Culture Collection (ATCC) on Oct. 3,1994 under the terms of the Budapest Treaty (for example,rV-CEA/_(n)IL-2 (ATCC Designation VR 2480), rV-_(m)B7-2 (ATCCDesignation VR 2482); and rV-_(m)B7-1 (ATCC Designation VR 2483)).

DNA sequences encoding a Mtb polypeptide can be expressed in vitro byDNA transfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

As noted above, a polynucleotide sequence encoding a Mtb polypeptide canbe operatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

Hosts cells can include microbial, yeast, insect and mammalian hostcells. Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Non-limitingexamples of suitable host cells include bacteria, archea, insect, fungi(for example, yeast), mycobacterium (such as M. smegmatis), plant, andanimal cells (for example, mammalian cells, such as human). Exemplarycells of use include Escherichia coli, Bacillus subtilis, Saccharomycescerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells,Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.Techniques for the propagation of mammalian cells in culture arewell-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Methods inEnzymology, volume 58, Academic Press, Inc., Harcourt Brace Jovanovich,N.Y.). Examples of commonly used mammalian host cell lines are VERO andHeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although celllines may be used, such as cells designed to provide higher expressiondesirable glycosylation patterns, or other features. As discussed above,techniques for the transformation of yeast cells, such as polyethyleneglycol transformation, protoplast transformation and gene guns are alsoknown in the art (see Gietz and Woods Methods in Enzymology 350: 87-96,2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a Mtb polypeptide,and a second foreign DNA molecule encoding a selectable phenotype, suchas the herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein (see for example, Eukaryotic Viral Vectors, ColdSpring Harbor Laboratory, Gluzman ed., 1982).

Therapeutic Methods and Pharmaceutical Compositions

The Mtb polypeptides disclosed herein, or nucleic acids encoding the Mtbpolypeptides, can be used to generate an immune response in a subject.In several examples, the subject is infected with Mtb or is at risk ofbeing infected with Mtb. Thus, in several embodiments, the methodsinclude administering to a subject a therapeutically effective amount ofone or more of the Mtb polypeptides disclosed herein (or polynucleotidesencoding these polypeptides), in order to generate an immune response,such as, but not limited to, a protective immune response.

In exemplary applications, compositions are administered to a subjecthaving in an amount sufficient to produce an immune response to Mtb.These Mt polypeptides, or polynucleotides encoding these polypeptidesare of use to prevent an infection with Mtb, prevent progression todisease in a subject having a latent Mtb infection, or to treattuberculosis in a subject infected with Mtb. In several examples,administration of a therapeutically effective amount of a compositionincluding one or move of the Mtb polypeptides disclosed herein (orpolynucleotides encoding these polypeptides) induces a sufficient immuneresponse to decrease a symptom of a disease due to Mtb infection, toprevent the development of one or more symptoms of tuberculosis, or toprevent infection with Mtb.

In some examples, the compositions are of use in preventing a futureinfection with Mtb. Thus, a therapeutically effective amount of thecomposition is administered to a subject at risk of becoming infectivewith Mtb. The composition prevents the development of tuberculosis, suchas latent or active tuberculosis in the subject upon subsequent exposureto Mtb.

In additional examples, the compositions are administered to a subjectwith a, latent Mtb infection, and prevent the development of symptoms oftuberculosis. Thus the compositions are of use in treating a subjectwith latent tuberculosis, such that the subject does not develop activetuberculosis.

Amounts effective for these uses will depend upon the severity of thedisease, the general state of the patient's health, and the robustnessof the patient's immune system. In one example, a therapeuticallyeffective amount of the compound is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inother examples, a therapeutically effective amount is an amountsufficient to prevent an infection with Mtb in a subject upon subsequentexposure of the subject to Mtb. In additional examples, atherapeutically effective amount is an amount sufficient to preventdevelopment of symptom in a subject infected with Mtb.

A Mtb polypeptide can be administered by any means known to one of skillin the art (see Banga, A., “Parenteral Controlled Delivery ofTherapeutic Peptides and Proteins,” in Therapeutic Peptides andProteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) eitherlocally or systemically, such as by intramuscular injection,subcutaneous injection, intraperitoneal infection, intravenousinjection, oral administration, nasal administration, transdermaladministration or even anal administration. In one embodiment,administration is by oral, subcutaneous injection or intramuscularinjection. To extend the time during which the peptide or protein isavailable to stimulate a response, the peptide or protein can beprovided as an implant, an oily injection, or as a particulate system.The particulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle. (see, e.g., Banga,supra). A particulate carrier based on a synthetic polymer has beenshown to act as an adjuvant to enhance the immune response, in additionto providing a controlled release. Aluminum salts can also be used asadjuvants to produce an immune response.

In one specific, non-limiting example, the Mtb polypeptide isadministered in a manner to direct the immune response to a cellularresponse (that is, a cytotoxic T lymphocyte (CTL) response), rather thana humoral (antibody) response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF; one or more molecules such as OX-40L or 41 BBL, or combinationsof these molecules, can be used as biological adjuvants (see, forexample, Salgaller et al., 1998, J. Surg. Oncol. 68(2):122-38; Lotze etal., 2000, Cancer J. Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, StemCells 16(Suppl 1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol.465:381-90). These molecules can be administered systemically (orlocally) to the host. In several examples, IL-2, RANTES, GM-CSF, TNF-α,IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, B7-1, B7-2, OX-40L, 41 BBL andICAM-1 are administered.

A number of means for inducing cellular responses, both in vitro and invivo, are known. Lipids have been identified as agents capable ofassisting in priming CTL in vivo against various antigens. For example,as described in U.S. Pat. No. 5,662,907, palmitic acid residues can beattached to the alpha and epsilon amino groups of a lysine residue andthen linked (for example, via one or more linking residues, such asglycine, glycine-glycine, serine, serine-serine, or the like) to animmunogenic peptide. The lipidated peptide can then be injected directlyin a micellar form, incorporated in a liposome, or emulsified in anadjuvant. As another example, E. coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561, 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable. A pharmaceutical compositionincluding a Mtb polypeptide is thus provided.

These compositions are of use to promote an immune response to Mtb. Inone embodiment, the Mtb polypeptide is mixed with an adjuvant containingtwo or more of a stabilizing detergent, a micelle-forming agent, and anoil. Suitable stabilizing detergents, micelle-forming agents, and oilsare detailed in U.S. Pat. Nos. 5,585,103; 5,709,860; 5,270,202; and5,695,770, all of which are incorporated by reference. A stabilizingdetergent is any detergent that allows the components of the emulsion toremain as a stable emulsion. Such detergents include polysorbate, 80(TWEEN) (Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl;manufactured by ICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™,TWEEN 60™, ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. Thesedetergents are usually provided in an amount of approximately 0.05 to0.5%, such as at about 0.2%. A micelle forming agent is an agent whichis able to stabilize the emulsion formed with the other components suchthat a micelle-like structure is formed. Such agents generally causesome irritation at the site of injection in order to recruit macrophagesto enhance the cellular response. Examples of such agents includepolymer surfactants described by BASF Wyandotte publications, e.g.,Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J.Immunol. 129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, andTETRONIC™ 1501, 150R1, 701, 901, 1301, and 130R1. The chemicalstructures of such agents are well known in the art. In one embodiment,the agent is chosen to have a hydrophile-lipophile balance (HLB) ofbetween 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167,1984. The agent can be provided in an effective amount, for examplebetween 0.5 and 10%, or in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, such as to provide a vehiclefor the desired antigen, and preferably has a melting temperature ofless than 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, or between 2.5 and 5%.The oil should be both biodegradable and biocompatible so that the bodycan break down the oil over time, and so that no adverse affects, suchas granulomas, are evident upon use of the oil.

In one embodiment, the adjuvant is a mixture of stabilizing detergents,micelle-forming agent, and oil available under the name PROVAX® (IDECPharmaceuticals, San Diego, Calif.). An adjuvant can also be animmunostimulatory nucleic acid, such as a nucleic acid including a CpGmotif, or a biological adjuvant (see above).

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems, see Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., 1995. Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres, the therapeutic agent is dispersedthroughout the particle. Particles, microspheres, and microcapsulessmaller than about 1 μm are generally referred to as nanoparticles,nanospheres, and nanocapsules, respectively. Capillaries have a diameterof approximately 5 μm so that only nanoparticles are administeredintravenously. Microparticles are typically around 100 μm in diameterand are administered subcutaneously or intramuscularly (see Kreuter,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; and5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding an Mtb polypeptide. A therapeutically effective amount ofthe Mtb polynucleotide can be administered to a subject in order togenerate an immune response.

Optionally, one or more cytokines, such as IL-2, IL-6, IL-12, RANTES,GM-CSF, TNF-α, or IFN-γ, one or more growth factors, such as GM-CSF orG-CSF, one or more costimulatory molecules, such as ICAM-1, LFA-3, CD72,B7-1, B7-2, or other B7 related molecules; one or more molecules such asOX-40L or 41 BBL, or combinations of these molecules, can be used asbiological adjuvants (see, for example, Salgaller et al., 1998, J. Surg.Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J. Sci. Am. 6(Suppl1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl 1):251-60; Kuiper etal., 2000, Adv. Exp. Med. Biol. 465:381-90). These molecules can beadministered systemically to the host. It should be noted that thesemolecules can be co-administered via insertion of a nucleic acidencoding the molecules into a vector, for example, a recombinant poxvector (see, for example, U.S. Pat. No. 6,045,802). In variousembodiments, the nucleic acid encoding the biological adjuvant can becloned into same vector as the Mtb polypeptide coding sequence, or thenucleic acid can be cloned into one or more separate vectors forco-administration. In addition, nonspecific immunomodulating factorssuch as Bacillus Cahnette-Guerin (BCG) and levamisole can beco-administered.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding an Mtb polypeptide canbe placed under the control of a promoter to increase expression of themolecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response), and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce Class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, an Mtbpolypeptide can also be expressed by attenuated viral hosts or vectorsor bacterial vectors. Recombinant vaccinia virus, adeno-associated virus(AAV), herpes virus, retrovirus, or other viral vectors can be used toexpress the peptide or protein, thereby eliciting a CTL response. Forexample, vaccinia vectors and methods useful in immunization protocolsare described in U.S. Pat. No. 4,722,848. BCG (Bacillus Calmette Guerin)provides another vector for expression of the peptides (see Stover,Nature 351:456-460, 1991).

When a viral vector is utilized, it is desirable to provide therecipient with a dosage of each recombinant virus in the composition inthe range of from about 10⁵ to about 10¹⁰ plaque forming units/mgmammal, although a lower or higher dose can be administered. Thecomposition of recombinant viral vectors can be introduced into a mammal(1) prior to any evidence of an infection with Mtb; (2) to preventdevelopment of tuberculosis in an individual infected with tuberculosis;or (3) to decrease a symptom of tuberculosis in a mammal infected withMtb. Examples of methods for administering the composition into mammalsinclude, but are not limited to, exposure of cells to the recombinantvirus ex vivo, or injection of the composition into the affected tissueor intravenous, subcutaneous, intradermal or intramuscularadministration of the virus. Alternatively the recombinant viral vectoror combination of recombinant viral vectors may be administered locallyin a pharmaceutically acceptable carrier. Generally, the quantity ofrecombinant viral vector, carrying the nucleic acid sequence of one ormore Mtb polypeptides to be administered is based on the titer of virusparticles. An exemplary range of the immunogen to be administered is 10⁵to 10¹⁰ virus particles per mammal, such as a human.

In the embodiment where a combination of a first recombinant viralvector carrying a nucleic acid sequence of one or more Mtb polypeptideand a second recombinant viral vector carrying the nucleic acid sequenceof one or more immunostimulatory molecules is used, the mammal can beimmunized with different ratios of the first and second recombinantviral vector. In one embodiment the ratio of the first vector to thesecond vector is about 1:1, or about 1:3, or about 1:5. Optimal ratiosof the first vector to the second vector may easily be titered using themethods known in the art (see, for example, U.S. Pat. No. 6,893,869,incorporated herein by reference).

In one embodiment the recombinant viruses have been constructed toexpress cytokines (such as TNF-α, IL-6, GM-CSF, and IL-2), andco-stimulatory and accessory molecules (B7-1, B7-2) alone and in avariety of combinations. Simultaneous production of an immunostimulatorymolecule and the Mtb polypeptide enhances the generation of specificeffectors. Without being bound by theory, dependent upon the specificimmunostimulatory molecules, different mechanisms might be responsiblefor the enhanced immunogenicity: augmentation of help signal (IL-2),recruitment of professional APC (GM-CSF), increase in CTL frequency(IL-2), effect on antigen processing pathway and MHC expression (IFNγand TNFα) and the like. For example, IL-2, IL-6, interferon, tumornecrosis factor, or a nucleic acid encoding these molecules, can beadministered in conjunction with a Mtb immunogenic polypeptide, or anucleic acid encoding an Mtb polypeptide. The co-expression of a Mtbpolypeptide together with at least one immunostimulatory molecule can beeffective in an animal model to show anti-tumor effects.

In one embodiment, a nucleic acid encoding an Mtb polypeptide isintroduced directly into cells. For example, the nucleic acid can beloaded onto gold microspheres by standard methods and introduced intothe skin by a device such as Bio-Rad's Helios™ Gene Gun. The nucleicacids can be “naked,” consisting of plasmids under control of a strongpromoter. Typically, the DNA is injected into muscle, although it canalso be injected directly into other sites, including tissues inproximity to metastases. Dosages for injection are usually around 0.5μg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5mg/kg (see, for example, U.S. Pat. No. 5,589,466).

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration would include about 0.1 μg to 10 mg ofimmunogenic Mtb polypeptide per patient per day. Dosages from 0.1 up toabout 100 mg per patient per day can be used, particularly if the agentis administered to a secluded site and not into the circulatory or lymphsystem, such as into a body cavity or into a lumen of an organ. Actualmethods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed.,Mack Publishing Company, Easton, Pa., 1995.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thesubject. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until atherapeutic result is achieved. In one embodiment, the dose issufficient to treat or ameliorate symptoms or signs of tuberculosiswithout producing unacceptable toxicity to the subject. In anotherembodiment, the dose is sufficient to prevent infection with Mtb uponsubsequent exposure to Mtb. In a further embodiment, the dose issufficient to prevent a symptom of tuberculosis in a subject with alatent Mtb infection. Systemic or local administration can be utilized.

In another method, antigen presenting cells (APCs), such as dendriticcells, are isolated from a subject of interest and pulsed orco-incubated with peptides comprising an Mtb polypeptide in vitro. Inone specific, non-limiting example, the antigen presenting cells areautologous cells isolated from the subject of interest. Atherapeutically effective amount of the antigen presenting cells isadministered (re-introduced) to the subject of interest.

The Mtb polypeptide can be delivered to the dendritic cells or todendritic cell precursors via any method known in the art, including,but not limited to, pulsing dendritic cells directly with antigen, orutilizing a broad variety of antigen delivery vehicles, such as, forexample, liposomes, or other vectors known to deliver antigen to cells.In one specific, non-limiting example an antigenic formulation includesabout 0.1 μg to about 1,000 μg, or about 1 to about 100 μg of a selectedMtb polypeptide. The Mtb polypeptide can also be administered withagents that promote dendritic cell maturation. Specific, non-limitingexamples of agents of use are interleukin-4 (IL-4) andgranulocyte/macrophage colony stimulating factor (GM-CSF), or flt-3ligand (flt-3L). The preparation can also contain buffers, excipients,and preservatives, amongst other ingredients.

In one embodiment, mature antigen presenting cells are generated topresent the immunogenic Mtb polypeptide. These dendritic cells are thenadministered alone to a subject infected with Mtb, or at risk forinfection with Mtb. In another embodiment, the mature dendritic cellsare administered in conjunction with an antiviral agent.

Alternatively, the APCs are used to sensitize CD8 cells, such asperipheral blood lymphocytes (PBLs). The PBLs can be from the samesubject (autologous) that is to be treated. Alternatively, the PBLs canbe heterologous. However, they should at least be MHC Class-I restrictedto the HLA types the subject possesses. An effective amount of thesensitized cells are then administered to the subject.

Peripheral blood mononuclear cells (PBMCs) can be used as the respondercell source of cytotoxic T lymphocyte (CTL) precursors. The appropriateantigen-presenting cells are incubated with peptide, after which thepeptide-loaded antigen-presenting cells are then incubated with theresponder cell population under optimized culture conditions. PositiveCTL activation can be determined by assaying the culture for thepresence of CTLs that kill radio-labeled target cells, both specificpeptide-pulsed targets as well as target cells expressing endogenouslyprocessed forms of the antigen from which the peptide sequence wasderived.

Alternatively, a CD8⁺ T cell clone that recognizes the Mtb polypeptidecan be isolated from a subject of interest. This CD8⁺ T cell clone canbe expanded in vitro, using methods known in the art. A therapeuticallyeffective amount of the CD8⁺ T cells is then administered to the subjectof interest.

Thus, cells can be administered to a subject to treat or an Mtbinfection, such as to decrease a symptom of an Mtb infection. In theseapplications, a therapeutically effective amount of activated antigenpresenting cells, or activated lymphocytes, are administered to asubject in an amount sufficient to raise an immune response to Mtb.

In supplemental methods, any therapeutic regimen is augmented byadministering a cytokine, such as interleukin (IL)-2, IL-3, IL-6, IL-10,IL-12, IL-15, GM-CSF, interferons. In further methods, an additionalantiviral agent is administered to the subject.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

For many infections, the repertoire of the CD8 response is shaped by theentry of antigen into the MHC-I processing pathway, binding of peptidesand/or non-peptide antigens to MHC-I molecules, and recognition of thesestructures by T cells. Ultimately, a relatively limited subset ofpathogen-specific T cells emerge. While a number of commonly recognizedCD4 Mtb antigens have been described (Reed et al., Microbes Infect7:922-931, 2005) (ESAT-6, CFP10, Ag85, etc.), surprisingly little isknown about common Mtb antigens recognized by human CD8⁺ T cells. Themajority of CD8 epitopes that have been identified were defined bytesting of Mtb peptides selected for high affinity binding to MHC ClassIa molecules (HLA-A2 in most cases (see, for example, Lalvani, MicrobesInfect 7:922-931, 1998)). In almost all of these, however, the ex vivofrequency of these T cells in Mtb-infected individuals is low orundetectable, suggesting that these specificities may not representimmunodominant responses. In contrast, in the limited cases in which Tcells have been used to define epitopes contained in selected Mtbantigens, high ex vivo frequencies have been demonstrated (see Lewinsohnet al., Am J Respir Crit Care Med 166:843-848, 2002), suggesting, that aT cell-centered approach can identify immunodominant epitopes. Moreover,CD8 T cell responses to some Mtb antigens which represent good CD4antigens (CFP10, ESAT-6, Ag85, and Mtb39) have been detected at highfrequency in persons infected with Mtb. Therefore, a limited library ofoverlapping synthetic peptides representing several known CD4 Mtbantigens was used to determine the magnitude of the CD8 response tothese antigens in persons with active tuberculosis (TB) and latenttuberculosis infection (LTBI) as well as uninfected subjects.Furthermore, a panel of Mtb-specific CD8⁺ T cell clones was utilized todefine minimal epitopes recognized within these antigens and determinedthe contribution of these novel epitopes to the ex vivo Mtb-specific CD8response.

Example 1 Materials and Methods

Human subjects. Uninfected individuals were defined as healthyindividuals with a negative tuberculin skin test (TST) and no know riskfactors for infection with Mtb. Individuals with LTBI were defined ashealthy persons with a positive TST and no symptoms and signs of activeTB. In all active TB cases, pulmonary TB was diagnosed by the TBController of the county and confirmed by positive sputum culture forMycobacterium tuberculosis. Peripheral blood mononuclear cells (PBMC)were isolated from whole blood obtained by venipuncture or apheresis.

Media and Reagents. Culture medium consisted of RPMI 1640 supplementedwith 10% Fetal Bovine Sera (FBS; Bio Whittaker), 5×10⁻⁵ M 2 ME(Sigma-Aldrich), and 2 mM glutamine (GIBCO BRL). For the growth andassay of Mtb-reactive T cell clones, RPMI 1640 was supplemented with 10%human serum. Mtb strain H37Rv was obtained from the American TypeCulture Collection (10801 Universtity Bouleveard., Manassas, VA20110-2209) and prepared as previously described (Lewinsohn et al., JImmunol 165:925-930, 2000). Peptides were synthesized by GenemedSynthesis, Inc, (San Francisco, Calif.). Synthetic peptide poolsconsisted of 15-mers overlapping by 11 amino acids (aa) representing Mtbproteins demonstrated to be potent CD4 antigens. Peptide poolsrepresenting CFP-10 (Berthet et al., Microbiology 144:3195-3203, 1998;Dillon et al., J Clin Microbiol 38:3285-3290, 2000), ESAT-6 (Sorenson etal., Infect Immun 63:1710-1717, 1995), Mtb39a (two pools, A & B,reference) (Dillon et al., Infect Immun 67:2941-2950, 1999), Mtb8.4(Coler et al., J Immunol 161:2356-2364, 1998), Mtb 9.9 (Alderson et al.,J Exp Med 191:551-560, 2000), (Coler et al., J Immunol 161:2356-2364,1998), Mtb 9.9 (Alderson et al., J Exp Med 191:551-560, 2000), EsxG(Rosenkrands et al., Electrophoresis 21:3740-3756, 2002), 19 kDa antigen(Collins et al. J Gen Microbiol 136:1429-1436, 1990), antigen 85 b(Borremans et al., Infect Immun 57:3123-3130, 1989) (two pools, A & B,reference) were synthesized. Peptides were resuspended in DMSO and up to50 peptides were combined into one pool such that each peptide in thepool was at a concentration of lmg/ml. Peptide pools were stored at −80°C.

Cell Lines and T Cell Clones. EBV-transformed B cell lines, LCL, wereeither generated using supernatants from the cell line 9B5-8 (AmericanType Culture Collection, Manassas, Va.) or obtained from the NationalMarrow Donor Program (NMDP; Minneapolis, MN). LCL were maintained bycontinuous passage as previously described (Heinzel et al., J Exp Med196:1473-1481, 2002). Mtb-specific T cell clones were isolated fromindividuals with LTBI or active tuberculosis, using Mtb-infected DCs asAPCs and limiting dilution cloning methodology as previously described(Lewinsohn et al., J Immunol 165:925-930, 2000). Briefly, CD8⁺ T cellswere isolated from PBMC using negative selection using CD4antibody-coated beads and then positive selection using CD8antibody-coated magnetic beads per the manufacturer's instructions(Miltenyi Biotec, Auburn Calif.) or via flow cytometry. In this case,CD4-PE (BD Biosciences cat# 555347) negative, CD8-APC (BD Biosciences,cat# 555369) positive cells (purity >99%) were sorted on a BectonDickenson LSR II. T cells were seeded at various concentrations in thepresence of a 1×10⁵ irradiated autologous Mtb-infected DC, generated asdescribed below, and rIL-2 (5 ng/ml) in cell culture media consisting of200 μl of RPMI 1640 supplemented with 10% human sera. Wells exhibitinggrowth between 10-14 days, were assessed for Mtb specificity usingELISPOT and Mtb-infected DC as a source of APCs. T cells retaining Mtbspecificity were further phenotyped for αβ T cell receptor expressionand CD8 expression by FACS and expanded as described below. Vβ usage wasdetermined using the IOTest Beta Mark Kit from Beckman Coulter.

Expansion of T cell clones. To expand the CD8⁺ T cell clones, a rapidexpansion protocol using anti-CD3 mAb stimulation was used as describedpreviously (Heinzel et al., J Exp Med 196:1473-1481, 2002).

Generation and Infection of Peripheral Blood DCs. Monocyte-derived DCswere prepared (Heinzel et al., supra; Romani et al., J Exp Med180:83-93, 1994). To generate Mtb-infected DC, cells (1×10⁶) werecultured overnight in the presence of Mtb (multiplicity of infection[MOI]=50:1). After 18 hours, the cells were harvested and resuspended inRPMI/10% human serum.

MHC binding assays. The MHC-peptide binding assay utilized measures theability of peptide ligands to inhibit the binding of a radiolabeledpeptide to purified MHC molecules, and has been described in detailelsewhere (Sidney et al., 1999. UNIT 18.3 Measurement of MHC/peptideinteractions by gel filtration. In Current Protocols in Immunology.Coligan et al., eds., John Wiley & Sons, Inc., 1996). Briefly, purifiedMHC molecules, test peptides, and a radiolabeled probe peptide wereincubated at room temperature in the presence of human B2-microglobulinand a cocktail of protease inhibitors. After a two-day incubation,binding of the radiolabeled peptide to the corresponding MHC class 1molecule was determined by capturing MHC/peptide complexes on W6/32antibody (anti-HLA A, B, and C) or B123.2 (anti-HLA B, C and some A)coated plates, and bound counts per minute (cpm) were measured using amicroscintillation counter. For competition assays, the concentration ofpeptide yielding 50% inhibition of the binding of the radiolabeledpeptide was calculated. Peptides were typically tested at six differentconcentrations covering a 100,000-fold dose range, and in three or moreindependent assays. Under the conditions utilized, where [label]<[MHC]and IC₅₀≧[MHC], the measured IC₅₀ values are reasonable approximationsof the true Kd values.

IFN-γ ELISPOT assay. The IFN-γ ELISPOT assay was performed as describedpreviously (Beckman et al., J Immunol 157:2795-2803, 1996). Fordetermination of ex vivo frequencies of CD4⁺ or CD8⁺ T cells respondingto Mtb infection or Mtb antigens, CD4⁺ or CD8⁺ T-cells were positivelyselected from PBMC using magnetic beads (Miltenyi Biotec, Auburn Calif.)as a source of responder T cells and tested in duplicate at fourdifferent cell concentrations. Autologous DC (20,000 cells/well) wereused as APC and DC were either infected with Mtb or pulsed with peptidepools (5 μg/ml, final concentration of each peptide) and then added tothe assay. For assays using T cell clones, T cells (1000 or 5000cells/well) were incubated with autologous LCL (20,000 cells/well) inthe presence or absence of antigen.

Data analysis: To determine the ex vivo frequency of antigen-specific Tcells, the average number of spots per well for each duplicate wasplotted against the number of responder cells per well. Linearregression analysis was used to determine the slope of the line, whichrepresents the frequency of antigen-specific T cells. The assay isconsidered positive, i.e. reflecting the presence of a primed T cellresponse, if the binomial probability (Lewinshon et al., Microbes Infect8:2587-2598, 2006) for the number of spots is significantly different byexperimental and control assays. To determine differences in ex vivo Tcell frequencies between groups, Wilcoxon/Kruskal-Wallis analysis wasused.

Example 2 Defining Immunodominant Mtb-Specific CD8+ Antigens

To define immunodominant Mtb-specific CD8⁺ antigens, and to determinewhether or not these responses result from infection with Mtb, CD8⁺ Tcells were used from donors uninfected, with LTBI, or actively infectedwith Mtb. Responses were determined either directly ex vivo, or usingCD8⁺ T cell clones obtained by limiting dilution cloning on Mtb-infectedautologous DC (Lewinsohn et al., J Immunol 165:925-930, 2000). As muchis known about dominant CD4⁺ Mtb antigens, a panel of these commonlyrecognized antigens was selected for further evaluation. These were:Mtb39, CFP10, and Mtb8.4, Mtb9.9, ESAT-6, Ag85b, 19 kDa, and EsxG. Toavoid bias introduced by using peptides of predicted HLA-bindingspecificity, we synthesized overlapping peptides (15 aa, overlap 11 aa)to represent the proteins of interest (Lewinshon et al., J Immunol166:439-446, 2001).

To accurately determine the ex vivo effector cell frequencies of CD8⁺ Tcells, linear regression analysis was used. As shown in FIG. 1, magneticbead purified CD8⁺ T cells were tested against peptide pulsed DC over arange of CD8⁺ T cell numbers in an IFN-γ ELISPOT assay. A positive assaywas determined as described below and if positive, the antigen specificfrequency was determined using linear regression.

Subjects uninfected (n=14), those with LTBI (n=20) and those with activeTB (n=12) were evaluated for CD8⁺ responses to a panel of Mtb CD4⁺ Tcell antigens, as well as to Mtb-infected DC. All subjects tested hadrobust CD8⁺ T cell responses to Mtb-infected DC and were of greatermagnitude in individuals with active TB than in those with LTBI (p=0.01;FIG. 2, Table I). However, CD8⁺ T cell responses to the panel of Mtbantigens were found almost exclusively in those infected with Mtb inthat statistically significant differences between uninfected andMtb-infected individuals were noted for seven of ten antigens for boththe magnitude of the response (FIG. 2) and the proportion of positiveassays (Table I).

TABLE I CD8⁺ T cell responses to known TB antigens. Mtb Infected MtbUninfected Antigen # positive^(a)/# tested (%) # positive^(a)/# tested(%) P value (2 tail fishers) Mtb DC 17/17 (100) 11/11 (100) Mtb39 Pool A13/30 (43)   0/14 (0)  0.003 Mtb 39 Pool B 10/30 (33)   0/14 (0)  0.01CFP10 14/30 (47)   1/14 (7)  0.02 Mtb 8.4 13/30 (43)   0/14 (0)  0.003Mtb 9.9 10/25 (40)   1/14 (7)  0.06 ESAT 6 12/25 (48)   0/14 (0)  0.003Ag85b Pool A  5/22 (23)   1/14 (7)  0.37 Ag85b Pool B  4/22 (18)   0/14(0)  0.14 19 kd  6/22 (27)   1/12 (8)  0.38 EsxG  9/22(41)    0/14 (0) 0.006 ^(a)Positive assay defined in text.

However differences in CD8⁺ T cell responses between individuals withactive TB and LTBI were not statistically different. While strong CD8⁺ Tcell responses were observed against many of the antigens tested, it isequally notable that several subjects with strong Mtb directed CD8⁺ Tcell responses did not have demonstrable responses to many of theantigens tested.

These ex vivo frequency data demonstrate the presence of high-frequencyresponses to a number of known Mtb antigens, but do not shed light onthe restricting allele, minimal epitope, or dominance hierarchy withinthe gene of interest. To address this question, limiting dilutioncloning of human CD8⁺ T cells using Mtb-infected DC was performed (seeLewinsohn et al., J Immunol 166:439-446, 2001), and panels of bothclassically and non-classically HLA-restricted CD8⁺ T cell clones weregenerated. Using peptide pools representing known CD4⁺ antigens, theantigenic specificity of the HLA-Ia restricted clones can be defined inmore than half of the clones (Table II).

TABLE II Many CD8⁺ T cell clones recognize known CD4⁺ T cell antigensHLA-Ia Antigen # Distinct # Distinct Clones Identified Antigens EpitopesDonor Tb Status (#)^(a) (#)^(b) (#)^(c) (#)^(d) D431 Active TB 1 0 0 0D432 Active TB 14 4 2 2 D466 Active TB 11 10 1 2 D571 Active TB 7 7 1 1D480 Active TB 6 6 1 1 D481 Active TB 11 11 1 1 D426 LTBI 1 0 0 0 D443LTBI 1 1 1 1 D454 LTBI 2 2 2 2 D504 LTBI 7 1 1 1 Totals 61 42 10 11^(a)Number of clones derived from donor. ^(b)Number of clones for whichcognate antigen was identified. ^(c)Total number of distinct antigensidentifed from the clone set. ^(d)Total number of distinct epitopesidentified from the clone set.

This approach is demonstrated in detail for a single representativeclone, D466 D6, derived from a subject with active TB. As shown in FIG.3A, testing the clone against autologous DC pulsed with a panel ofpeptide pools unambiguously defined the antigenic specificity as CFP10.The clone was then tested against each of the 15-mer peptides thatcomprise the CFP10 pool, revealing that the epitope was contained withinCFP10₁₋₁₅ (FIG. 3B). Each possible 8 aa, 9 aa, 10 aa, and 11 aa peptidewas then synthesized and tested for reactivity, revealing antigenicactivity between aa 2-11 (FIG. 3C). Similarly, each clone was testedagainst lymphoblastoid cell lines (LCL) sharing at least one HLA-typewith the donor (FIG. 3D). Autologous LCL and IHW 9058 LCL, which shareB4501 and C1601, present the epitope to the clone, identifying bothB4501 and C1601 as possible restricting alleles. However, C1601⁺ D433LCL do not present the epitope, eliminating C1601 as a candidaterestricting allele. Therefore D466 D6 is restricted by HLA-B4501. Asdemonstrated in FIG. 4, by testing each plausible epitope over a broadrange of concentrations, the minimal epitope was defined as CFP10₂₋₁₀for D466 D6. Experimental data supporting the assignment of the minimalepitope is provided for each clone in the supplemental Figure. A summaryof the antigenic specificity, minimal epitope, and HLA-restrictingallele is presented in Table III. Unexpectedly, all but one of the Tcell clones were restricted by HLA-B alleles. Furthermore, a minority ofthose observed were 9 aa in length.

TABLE III Summary of Epitopes Identified Epitope HLA- Sequence MHC VAccession Restrict Epitope (SEQ ID  Bind. beta Clone^(a) Gene NumberAllele Locat'n NO: 26-38) #SFU^(b) Aff^(c) region D160 1-IB^(d) (0)CFP10 Rv3874 B44  2-11 AEMKTDAATL  360  38 D160 1-6F^(d) (0) CFP10Rv3874 B14 85-94 RADEEQQQAL  120 NA D432 H12 (2) CFP10 Rv3874 B351449-58 TAAQAAVVRF  258 2011^(e)  5.3 D466 A10 (10) CFP10 Rv3874 B4501 2-9AEMKTDAA 2458   48 IND D466 D6 (1) CFP10 Rv3874 B4501  2-12 AEMKTDAATLA1993   6.2 22 D481 C10 (10) CFP10 Rv3874 B1502 75-83 NIRQAGVQY 1715 14^(f)  9 D481 C11 (1) CFP10 Rv3874 B1502 75-83 NIRQAGVQY 1715  14^(f)13.6 D480 F6 (6) CFP10 Rv3874 B0801  3-11 EMKTDAATL  387  79 13.1D571 B12 (3) CFP10 Rv3874 B4402  2-11 AEMKTDAATL   31  38 INDD571 E9 (4) CFP10 Rv3874 B4402  2-11 AEMKTDAATL   31  38 14 D504 E4 (1)Mtb9. Rv0287 A0201  3-11 LLDAHIPQL  <10   0.39  8 8 D454 B10 (1) Mtb9.Rv0287 B0801 53-61 AAHARFVAA   88   0.22 IND 8 D454 H1-2 (1) Mtb8.Rv1174c B1501  5-15 AVINTTCNYGQ   24  10  7.1 4 D432 A3 (2) Mtb Rv1174cB3514 32-40 ASPVAQSYL  210 127^(e) 14 8.4 D443 H9 (1) Ag85 Rv1886c TBD144-153 ELPQWLSANR  <10 NA 22 B ^(a)Number of sister clones is inparentheses. ^(b)# of SFU/250,000 CD8⁺ T cells is shown. ^(c)IC50 in nmis shown. ^(d)Published previously J Immunol. 2001 Jan 1; 166(1):439-46. ^(e)Measured binding affinity to B3501 is shown. ^(f)Measuredbinding affinity to B1501 is shown. NA = Not Available. IND =Indeterminate TBD = To be done.

Because each of the individual CD8⁺ T cell clones were derived based ongrowth of Mtb-infected DC, it was determined whether or not the antigenand epitopes identified reflected immunodominant epitopes ex vivo. Twoindependent approaches were pursued, the first to determine if theresponse was present at high frequency, and the second to determine whatproportion of the total response to the antigen is constituted by theepitope. To determine the ex-vivo effector cell frequency, as describedin FIG. 1, each epitope was tested using autologous DC and magnetic beadpurified CD8⁺ T cells derived from the donor from whom the T cell cloneswas isolated. A summary of the effector cell frequencies is presented inTable III. For the majority, the epitopes reflect high frequencyresponses, and thus could be considered a response that has been primedby exposure to Mtb. Notably, T cell clones isolated from four donorsrecognized CFP10. To determine if the epitopes defined reflected asubstantial proportion of the total response to the antigen of interest,magnetic bead purified CD8⁺ T cells from three donors with sufficientavailable peripheral blood mononuclear cells (PBMC) were tested forreactivity to each individual 15-mer peptide, the peptide pool, andpeptide representing the minimal epitope. As is demonstrated in FIG. 5,the ex vivo frequencies to the minimal epitope, 15-mer peptide(s)containing the minimal epitope, and peptide pool were remarkablyconcordant. These data suggested that for each donor a dominancehierarchy has been clearly established, and is reflected in the originalclones. Finally, as is noted in Table III, daughter clones of identicalspecificity were frequently identified, a result that would be predictedbased on an immundominance hierarchy. TCR V beta staining was used toconfirm the clonal relationship between daughter clones. Interestingly,in two cases, the identical minimal epitope and HLA-restriction wasrepresented by two distinct clones (Table III).

Because much work on human CD8⁺ T cell responses to Mtb has relied uponthe use of HLA-prediction algorithms, as each epitope was defined weasked whether or not the epitopes would have been predicted by theseapproaches. Many of these epitopes were not ranked strongly. This mighthighlight the limitations of those algorithms at the time they wereused. To address this question experimentally, the IC₅₀ for each peptidethat had been synthesized in the course of definition of the minimalepitope was determined against a panel of human HLA molecules. Shown inTable III is the IC₅₀ for the minimal epitope with the cognaterestricting allele. The data demonstrated that the T cell epitopes boundavidly to HLA, and show a high degree of concordance between the T cellepitope data and HLA-binding data.

The data demonstrated that CD8⁺ T cell responses are present in personsinfected with Mtb at frequencies that are comparable to that seenfollowing many common viral infections such as vaccinia, influenza, andCMV. All but one of the epitopes that were mapped were restricted byHLA-B molecules. The data suggest that by using a T cell driven approachto epitope identification, dominant epitopes can be defined in humansinfected with Mtb.

Example 3 Screening of T Cell Clones Against a Genomic Peptide Library

The classically-restricted and non-classically-restricted T cell clones(see Table II above) that did not recognize one of the known Mtb antigenpeptide pools (Rv3875, Rv3874, Rv1886c, Rv0287, Rv3763, Rv1174c, Rv1196,Rv1793, Rv2346c, Rv1037c, Rv3619c and Rv1198) were screened against agenomic peptide library. This peptide library represents 389 genes,representing roughly 10% of the Mtb genome. The peptides are 15mersoverlapping by 11 for each gene product. 50 nmol of each peptide wassynthesized individually and then pooled into 777 pools of 50 peptidesin a 96 well format (nine plates). Five blank wells and one well of anirrelevant peptide pool, SW gag, were included on each of the nineplates. To screen the clones against the genomic peptide library, theclones are first expanded and tested against Mtb-infected DCs to ensurethat each clone from this particular expansion yields a robustMtb-specific signal in the ELISPOT assay. Then up to six T cell clonesare pooled. For the screen, T cell clones (5,000 cells/well of eachclone), autologous DCs (20,000 cells/well), IL-2 (0.5 ng/ml) and thepeptide pools (5 ug/ml, individual peptides) were incubated overnight at37 C in the ELISPOT assay. Only one technical replicate is done per poolbecause 5000 T cell clones per well with a peptide antigen produced anoverwhelmingly positive response, resulting in a definitive result. Sixclassical clones from D504 were screened against the genomic peptidelibrary, leading to the discovery of a new epitope. This epitope wasfrom a family of four proteins that includes EsxJ, EsxW, EsxK and EsxP.These proteins share 98% homology and differ at only 3 amino acids.There is a fifth member of this family, EsxM (Rv1792), that was notincluded in the genomic peptide library.

The clones were screened against the individual fifteen-mers for thesepeptide pools. All six classical clones recognized EsxJ 21-35. This is aregion of EsxJ that is identical to the other four members of thisfamily. Next, 9, 10 and 11 mer peptides were made from this 15mer andscreened against each clone. The minimal epitope was determined to beEsxJ 24-34. In addition, the HLA restriction was found to be B5701.

Example 4 Additional Screening of T Cell Clones Against a GenomicPeptide Library

Eleven classical clones from D432B were screened against the genomicpeptide library described above. The antigen was determined for twoclones, which led to the identification of two novel epitopes,PE_PGRS42₄₇₋₅₅ and PE9₅₃₋₆₇. The minimal epitope for one clone wasdetermined to be PE_PGRS42₄₇₋₅₅ and the HLA restriction was found to beB3514. The minimal epitope for the other clone is not yet determined,but is contained in the 15mer PE9₅₃₋₆₇. The HLA restriction for thisclone was found to be B3905.

TABLE IV Detail of Novel Epitopes from Genomic Peptide Library Screens.#SFU/ MHC 250,000 Binding TCR V Accession Epitope CD8+ T- MHC- Affinitybeta Clone Gene Number Location Epitope cells Restriction (IC50 nm)region D504 Esx* Rv1038c 24-34 QTVEDE- 84 B5701 TBD Indeter- F9 (6)SEQ ID NO: ARRMW minate 2 D432 PE9 Rv1088 53-67 RLFNAN- TBD B3905 TBD 8D8 (1) SEQ ID NO: AEEYHA- 7 LSA D432 PE/PGR Rv2487c 47-55 VSAAIAG- TBDB3514 TBD 7.1 H8(1) S42 SEQ ID NO: LF 8 Numher of clones recognizingepitope from each donor in parentheses. *This is a family of proteinsthat have almost identical sequences. The family consists of Rv1038c,Rv1197, Rv2347, Rv3620c.

TABLE V Summary of Completed Clone Screens. # Non- # classical #classical # Classical Classical # positive # of clones clones TBavailable available wells in confirmed # novel epitope epitope NOT DonorStatus (screened) (screened) screen hits epitopes identified identified426 PPD+ 1 (1) 4 (4) 1 0 0 0 1 431 Active 1 (1) 1 (1)  1** 0 0 0 1 432Active 11 (11) 14 (7) 11  3 2 3 8 454 PPD+ 1* (0) 6 (4) 0 0 0 0 0 466Active 1 (1) 4 (4) 1 0 0 0 1 504 PPD+ 6 (6) 9 (9) 5 4 1 6 0 21 (20) 38(29) 18  7 3 9 11 *The classical clone from D454 did not recognize Mtbupon re-expansion and was not screened against library. **The classicalclones from 426 and 431 were screened together, so there was onepositive well between both clones.

Example 5 Screening of Ex Vivo CD8⁺ T-Cells Against a Genomic PeptideLibrary

CD8+ T-cells from a LTBI donor, D610 (SE Asian) were screened againstthe genomic peptide library described above. Each plate of the genomicpeptide library was screened in duplicate, for a total of 18 ELISPOTplates per screen. CD8⁺ T-cells were prepared from cryopreserved PBMC byCD8⁺ selection using magnetic bead separations. Resulting cellpopulations contained ≧96% CD8⁺ T cells. CD8⁺ T cells (250,000cells/well), autologous DCs (20,000 cells/well), and IL-2 (0.5 ng/ml)were added to peptide (final 5 ug/ml, individual peptides) in theELISPOT plates. Five media control wells are included on each plate. Foreach plate, the mean of these five wells was subtracted from each wellof that plate to normalize between plates. Each technical replicate oneach plate was then scored. A well was scored positive if the spotforming units (SFU), less the mean of the media wells, was greater thanor equal to ten and the SFU was greater than or equal to twice the meanof the media. (Hudgens et al., J. Immunol. Methods 288: 19-34, 2004).This donor responded to the four peptide wells containing EsxJ, EsxW,EsxK and EsxP. CD8+ T-cells were then screened against each 15mer fromthese peptide pools and found to respond only to EsxJ 21-35, the sameregion of EsxJ, EsxW, EsxK and EsxP that is described in example 3above.

Seven additional donors were screened against the genomic peptidelibrary. The top 10 responses are detailed in Table 7. The four peptidepools highlighted in yellow contain peptides from only one gene. Thesefour genes contain four novel epitopes.

TABLE 7 Top 10 Responses from Peptide Pool Screens of Seven Donors. SpotForming Units are for 250,000 CD8+ T-cells. Average Peptide Pool DonorSFU RvNumbers Represented in Wells Functional Category C09_1 D560 208.2Rv1860(50): cell wall and cell processes C12_4 D545 156.4 Rv0468(27):Rv0456c(23): lipid metabolism A04_3 D454 136 Rv0284(17): Rv0288(11):Rv0287(22) cell wall and cell processes B10_3 D560 112.3 Rv1273c(50):cell wall and cell processes E04_4 D560 78.2 Rv0152c(40): Rv0151c(10):PE/PPE G12_8 D560 77.4 Rv3478(18): Rv3507(32): PE/PPE E07_4 D525 76.8Rv0159c(50): PE/PPE A10_8 D560 70.4 Rv3136(47): Rv3144c(3): PE/PPE E11_8D560 66.4 Rv3350c(50): PE/PPE E08_9 D545 60.2 Rv1404(13): Rv2711(37):regulatory proteins

Example 6 Animal Models

In tuberculosis research, the mouse model has been used extensively tomodel various aspects of the disease. Mice can be infected by a varietyof routes, including intravenous, intraperitoneal and tracheal. Oneroute is aerosolization of the organism for respiratory infection. Themice are exposed to the aerosol in a chamber (wither whole body or noseonly infection). The dose of invention can be varied by manipulating theconcentration of Mtb in the nebulizer or time of exposure. A low doseinfection, such as about 50 colony forming units (CFU) via aerosolresults in a slow and steady increase in bacterial numbers in the lungs,generally reaching a peak in four weeks, which coincides with the peaknumber of T cells in the lungs. The initial period is considered theacute stage of infection. Following infection, there is a disseminationof bacteria to the mediastinal lymph nodes. T cell priming is generallydetectable between two and three weeks. After about four weeks thebacterial numbers stabilize, and there is a slow progressive pathologicresponse. This system is of use for modeling active infection. Thus, theabove-described polypeptides, or polynucleotides encoding thesepolypeptides, can be administered prior to infection. The ability of theMtb polypeptides (or polynucleotides encoding these polypeptides) toprevent infection is then assessed. Alternatively, the mice areadministered Mtb, and the ability of the Mtb polypeptide (orpolynucleotide encoding these polypeptides) to treat the Mtb infectionis monitored. The effectiveness of the Mtb polypeptides (orpolynucleotides) can be monitored by measuring the T cell response, suchas the number of CD8⁺ or CD4⁺ T cells, and/or measuring the bacterialnumbers, and/or evaluating the pathology.

Exemplary protocols are provided below (see also Repique et al., Infec.Immun. 70: 3318-3323, 2002, incorporated herein by reference for anadditional protocol):

A. Short Term Mouse Model:

C57BL/6 mice are vaccinated with a composition including one or more Mtbpolypeptide, or a polynucleotide encoding these one or morepolypeptides, according to the appropriate protocol and then rested for4 to 6 weeks. Immunized mice are infected with a low dose aerosol 50-100CFU) of virulent M. tuberculosis and protection is evaluated byassessing the number of viable bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of mice byhomogenizing the organs and plating serial 10-fold dilutions on 7H11agar plates. Plates are incubated for up to 21 days and the number ofcolony forming units per organ determined.

BCG vaccinated mice have approximately 1 Log 10 protection in their lungand spleen when compared to PBS-treated mice.

B. Short Term Guinea Pig Model

Out-bred Hartley guinea pigs are vaccinated with a composition includingone or more Mtb polypeptide, or a polynucleotide encoding these one ormore polypeptides and then rested for 8 to 10 weeks. Immunized guineapigs are infected with a low dose aerosol (10-30 CFU) of virulent M.tuberculosis and protection is evaluated by assessing the number ofviable bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of guinea pigs byhomogenizing the organs and plating serial 10-fold dilutions on 7H11agar plates. Plates are incubated for up to 21 days and the number ofcolony forming units per organ determined. Lung and spleen segments arealso taken for histological analyses.

BCG vaccinated guinea pigs have approximately 2-3 Log₁₀ protection intheir lung and spleen when compared to PBS-treated guinea pigs. Inaddition, BCG vaccinated guinea pigs have well defined granulomas whencompared to unvaccinated animals.

C. Long Term Guinea Pig Model

The guinea pig model is similar to the mouse model, but the experimentsare open-ended survival type and can last for as long as 2 years. Guineapigs develop ‘classical’ granulomas similar to humans with activetuberculosis (TB), and as lung tissue necrosis progresses, they begin tolose weight and die of TB similar to humans. The number of colonyforming units in the lungs and spleen can be assessed. Histologicalexamination can also be performed to determine the degree of lunginvolvement and tissue destruction. After low-dose aerosol exposure inthe guinea pig the number of organisms increases progressively duringthe first three weeks and then plateaus into a chronic state. During thelater stages of infection there is increased bacterial load in the lungand this is associated with a worsening pathological condition. Withouttreatment, there is a concomitant rise in both CD4 and CD8 T cells inthe lungs of infected guinea pigs.

Out-bred Hartley guinea pigs are vaccinated with the experimentalvaccine according to the appropriate protocol and then rested for 8 to10 weeks. Immunized guinea pigs are then infected with a low doseaerosol (10-30 CFU) of virulent M. tuberculosis. Guinea pigs are weighedweekly and monitored daily for signs of disease (such as increasedrespiration and failure to thrive). Unvaccinated guinea pigs succumb toinfection from 20 to 25 weeks post challenge, while BCG vaccinatedguinea pigs survive for 50 to 55 weeks post challenge.

At necropsy, the lung and spleen are assessed for the number of CFU andthe extent of pathology. The relative protection of the experimentalcomposition is compared to BCG vaccinated animals.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A method for producing an immune response to Mycobacteriumtuberculosis in a subject, comprising administering to the subject atherapeutically effective amount of an isolated polypeptide, wherein theisolated polypeptide comprises the amino acid sequence set forth as SEQID NO: 8 thereby inducing an immune response to Mycobacteriumtuberculosis.
 2. The method of claim 1, comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising the isolated polypeptide comprising the aminoacid sequence set forth as SEQ ID NO:
 8. 3. The method of claim 1,comprising administering to the subject a therapeutically effectiveamount of an isolated polypeptide consisting of the amino acid sequenceset forth as SEQ ID NO:
 8. 4. The method of claim 1, comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising an isolated polypeptide consistingof the amino acid sequence set forth as SEQ ID NO: 8 that specificallybinds MHC class I.
 5. The method of claim 1, further comprisingadministering to the subject a therapeutically effective amount of anadjuvant.
 6. The method of claim 1, wherein the subject is infected withMycobacterium tuberculosis (Mtb).
 7. The method of claim 1, wherein thesubject is at risk for infection with Mycobacterium tuberculosis (Mtb).8. The method of claim 1, wherein the subject has a latent infectionwith Mycobacterium tuberculosis (Mtb).
 9. The method of claim 1, whereinthe isolated polypeptide is covalently linked to a carrier.
 10. A methodfor treating a subject infected with Mycobacterium tuberculosis,comprising administering to the subject a therapeutically effectiveamount of an isolated polypeptide, wherein the isolated polypeptidecomprises the amino acid sequences set forth as SEQ ID NO: 8 therebytreating the subject infected with Mycobacterium tuberculosis.
 11. Themethod of claim 10, wherein the subject infected with Mycobacteriumtuberculosis does not have a symptom of tuberculosis.
 12. The method ofclaim 10, wherein the isolated polypeptide is covalently linked to acarrier.
 13. The method of claim 10, further comprising administering tothe subject a therapeutically effective amount of an adjuvant.
 14. Themethod of claim 10, comprising administering to the subject atherapeutically effective amount of a polypeptide consisting of theamino acid sequence set forth as SEQ ID NO:
 8. 15. The method of claim10, wherein the subject is at risk for infection with Mycobacteriumtuberculosis (Mtb).
 16. The method of claim 10, wherein the subject hasa latent infection with Mycobacterium tuberculosis (Mtb).
 17. The methodof claim 10, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising the isolatedpolypeptide comprising the amino acid sequence set forth as SEQ ID NO:8.
 18. The method of claim 10, comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositionconsisting of an isolated polypeptide comprising the amino acid sequenceset forth as SEQ ID NO: 8.